Synapses are specialized structures that connect nerve cells to one another and allow information to be transmitted between the cells. Synapses are essential for learning and storing memories. Many proteins that regulate how signals are transmitted at synapses have already been studied. In this manner, much has been learned about their function in learning and memory.

Cells can commit suicide by a process called apoptosis, also known as programmed cell death. Apoptosis is not only triggered in damaged cells but is also necessary for an organism to develop correctly. In fruit flies, the protein Drep-2 is a member of a family of proteins that degrade the DNA of cells that undergo apoptosis.

Andlauer et al. found no evidence that Drep-2 plays a role in apoptosis, but have now found Drep-2 at the synapses of the brain of the fruit fly Drosophila. Drep-2 could be observed in close proximity to another type of protein called metabotropic glutamate receptors. Metabotropic glutamate receptors and their signaling pathways are important for regulating certain changes to the synapses that mediate learning processes. Indeed, Andlauer et al. found that flies that have lost the gene that produces Drep-2 were unable to remember smells when these were paired with a punishment. Stimulating the regulatory glutamate receptors with drugs helped to overcome learning deficits that result from the lack of Drep-2.

Alterations in the production of a protein called FMRP cause fragile X syndrome in humans, the most common form of hereditary mental disability originating from a single gene defect. Flies lacking the FMRP protein show learning deficits that are very similar to the ones seen in flies that cannot produce Drep-2. However, Andlauer et al. observed that flies lacking both Drep-2 and FMRP can learn normally.

Exactly how Drep-2 works in synapses to help with memory formation remains to be discovered, although there are indications that it boosts the effects of signaling from the glutamate receptors and counteracts FMRP. Further research will be needed to establish whether the mammalian proteins related to Drep-2 perform similar roles in the brains of mammals.

Caspase family proteases regulate cellular pathways by cleavage of target proteins. They are best known for their roles during programmed cell death. One of their targets is the DNase Dff40/CAD, which degrades DNA during apoptosis (Enari et al., 1998). Dff40 is a member of the DNA fragmentation factor (Dff) family of proteins, characterized by CIDE-N domains that mediate protein–protein interactions (Wu et al., 2008). In Drosophila, four CIDE-N domain proteins were identified and named Dff related protein Drep-1 to Drep-4 (Inohara and Nuñez, 1999). Caspase-regulated Drep-4 is the ortholog of mammalian Dff40/CAD (Yokoyama et al., 2000). Drep-4 is inhibited by Drep-1, the ortholog of Dff45/ICAD, which is also cleaved by caspases (Mukae et al., 2000). The two other Drosophila family members, Drep-2 and -3, were suggested to be additional regulators of apoptosis, solely based on in vitro interactions (Inohara and Nuñez, 1999; Park and Park, 2012).

We show here that Drep-2, contrary to the expectations, is a novel synaptic protein. We have generated drep-2 mutants, which display learning and memory deficits. Further analyses suggest that Drep-2 regulates these processes by intersecting with metabotropic glutamate receptor signaling.

Drosophila encodes two Dff40/CAD-related proteins, Drep-4 and Drep-2 (Inohara and Nuñez, 1999; Yokoyama et al., 2000) (Figure 1—figure supplement 1). Both proteins are controlled by caspase-mediated cleavage of their inhibitors Drep-1 and Drep-3, respectively (Mukae et al., 2000; Park and Park, 2012, 2013). The roles of Drep-4 and -1 during apoptosis have been firmly established, yet the functions of Drep-2 and -3 have so far remained unknown (Inohara and Nuñez, 1999; Park and Park, 2012; Tan et al., 2012).

Drep-2 is a novel synaptic protein without a discernable role in the regulation of apoptosis

The gene coding for Drep-2 is located on chromosome IIR and contains five exons (Figure 1A). While the last two exons are used in all known isoforms, the first three exons are included alternatively within the four isoforms drep-2-RA to -RD.

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High-throughput RT-PCR data indicate a strong enrichment of both drep-2-RA and drep-3 transcripts in the central nervous system (CNS), while drep-4 and -1 are expressed ubiquitously (Graveley et al., 2011). We conducted in situ hybridization using drep-2-RA constructs in order to confirm that the expression of drep-2 is nervous system specific (Figure 1B). Next, we produced polyclonal antibodies against a fusion protein containing the C-terminal half of the protein, which is part of all isoforms (Drep-2^C-Term). Western blots from wild-type fly head extracts probed with Drep-2^C-Term showed a double band (Figure 1C) of the size expected for Drep-2 isoforms (McQuilton et al., 2012).

We generated drep-2 mutants by FLP-mediated excision between FRT site-bearing transposons to explore the function of Drep-2. The transposons P(XP)d00223 and PBac(RB)e04659 were used for the deletion allele drep-2^ex13 (Figure 1A). A second deletion allele, drep-2^ex27, was established using transposon lines PBac(RB)e02920 and PBac(RB)e04659. All drep-2 exons are deleted in homozygous drep-2^ex13 animals, while no other annotated transcription unit is affected. In the smaller intragenic deletion drep-2^ex27, all drep-2 exons apart from the very small (12 bp) first exon are eliminated. Both Drep-2^C-Term antibody bands were absent in head extracts of both mutants (Figure 1C), confirming the complete elimination of Drep-2 expression in these deletion alleles. Flies lacking drep-2 were viable and fertile but shorter-lived than the isogenic controls (Figure 1—figure supplement 2).

Subsequently, we used the Drep-2^C−Term antibody for wholemount immunostainings of Drosophila brains. The synaptic neuropil was strongly labeled throughout the brains of larvae (not shown) and adults (Figure 2). In both drep-2 mutants, Drep-2^C−Term staining in the CNS was completely abolished (drep-2^ex13: Figure 2A,B).

Figure 2

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As a Dff family member, Drep-2 has been suggested to be involved in the apoptotic regulation of DNA degradation (Inohara and Nuñez, 1999; Park and Park, 2012). However, neuronal cell bodies lacked Drep-2^C−Term staining (Figure 2). We produced synaptosome-like preparations by fractionation of the adult Drosophila CNS to biochemically confirm the association of Drep-2 with synapses (Owald et al., 2012; Depner et al., 2014). Drep-2 was strongly enriched in fractions containing synaptic membranes (Figure 3A). By contrast, no enrichment of Drep-2 could be observed in the nuclear fraction.

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Mutants lacking drep-2 showed normal exterior morphology, including their facet eyes (Figure 3B). Fly facet eyes are highly ordered structures, already displaying abnormal patterns in cases of moderate misregulation of apoptosis (Wolff and Ready, 1991; Song et al., 2000). The regular facet eyes of drep-2 mutants, therefore, argue against a major function of the protein in apoptosis. Wholemount brain stainings of wildtypes and drep-2^ex13 mutants also showed no apparent morphological differences between either genotype (Figure 2A,B; Figure 5—figure supplement 1). If apoptosis was, nevertheless, misregulated in the CNS of drep-2 mutants in vivo, an altered count of cell bodies should be expected in adult flies. Drep-2 staining was especially prominent at KC synapses in the mushroom body (MB) calyx of wildtypes (Figure 2C). We, therefore, quantified the numbers of cell bodies of a subset of MB-intrinsic neurons (KCs). No differences between drep-2 mutants and controls could be observed (Figure 3C).

Drep-2 was reported to degrade DNA in vitro (Park and Park, 2012). This supposed nuclease activity was observed if purified Drep-2 was incubated in vitro with plasmid DNA at a molar ratio of protein:DNA 80:1 (Park and Park, 2012). However, we (Figure 3D) and another previous report (Inohara and Nuñez, 1999) found no evidence of a nuclease activity of Drep-2, even at high concentrations. Instead, Drep-2 appeared to precipitate DNA, as evident by plasmid DNA no longer entering the agarose gel when incubated with Drep-2. This precipitation might have generated the previous impression that DNA is degraded in the presence of Drep-2. Taken together, we could not find evidence for an in vivo role of Drep-2 in regulating apoptosis in the CNS. While a function of the protein related to apoptosis can still not be fully excluded, we decided rather to examine the synaptic functions of Drep-2.

Drep-2 is strongly enriched at the postsynaptic densities of mushroom body input synapses

In order to narrow down a potential site of action of the protein, we examined Drep-2 expression in the adult CNS in more detail. While Drep-2^C-Term stained synapses throughout the brain, including optic lobes, antennal lobes, and the central complex (Figure 2), the immunoreactivity was particularly pronounced in the MB calyx (Figures 2C and Figure 4A).

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The principle circuitry processing olfactory information in Drosophila is highly similar to mammals (Davis, 2004). Signals are transferred in the fly's antennal lobe to projection neurons (PNs), which target the mushroom body (MB) calyx and the lateral protocerebrum. PNs synapse in the calyx onto MB-intrinsic Kenyon cells (KCs) by forming large cholinergic presynaptic boutons. These presynaptic specializations are tightly encircled by acetylcholine (ACh) receptor-expressing postsynaptic densities (PSDs) of KC dendritic claws (Yasuyama et al., 2002; Leiss et al., 2009a).

In the MB calyx, the Drep-2^C-Term signal overlapped with Dα7 ACh receptor subunits expressed in KC PSDs (Figure 4A,B,C′), which surrounded Bruchpilot (Brp)-positive (but Drep-2-negative) PN presynapses (Figure 4A,B, Figure 4—figure supplement 1A). Furthermore, Drep-2 colocalized with the postsynaptic protein Discs large, but clearly segregated from presynaptic choline acetyltransferase (Figure 4C). Consistent with endogenous Drep-2 being present at KC-derived PSDs, overexpression of UAS-Drep-2^mStrawberry with either pan-neural (elav^c155-Gal4) or KC-specific drivers (c305a-Gal4) resulted in an mStrawberry signal equivalent to that of the Drep-2^C-Term antibody (Figure 4—figure supplement 1B,C). Overexpression with a PN driver (gh146-Gal4), however, produced only a weak, diffuse expression pattern that bore no similarity to endogenous Drep-2 staining (Figure 4—figure supplement 1D). Moreover, re-expression of UAS-Drep-2 in KCs of drep-2 mutants produced a distinctive label at KC PSDs (Figure 4—figure supplement 1E). Finally, we confirmed the presence of Drep-2 at postsynaptic membranes of PN-KC synapses by immunoelectron microscopy (Figure 4D). Thus, Drep-2 accumulates at postsynaptic specializations of KCs within the MB calyx.

Drep-2 is required in KCs for normal olfactory short- and intermediate-term memory

Kenyon cells have an essential function in olfactory learning (Davis, 2011; Dubnau and Chiang, 2013). Here, conditioned stimuli (odors) get associated with unconditioned stimuli (e.g., electric foot shock). Because Drep-2 was strongly enriched at PSDs of KCs, we wondered whether the protein might contribute to olfactory learning. In aversive olfactory conditioning, flies are trained to learn the difference between a punished and an unpunished odor. We measured short-term memory (STM) directly following training and intermediate-term memory (ITM) after 3 hr. All flies used for behavioral experiments were outcrossed into our isogenic background (w¹¹¹⁸) for at least five generations. Performance of controls allowed for the detection of differences in either direction (Figure 5B,C).

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Mutants lacking drep-2 showed normal naïve sensory acuity, with innate olfactory responses and shock avoidance behavior not significantly different from controls (Figure 5A). However, the STM performance scores of both mutants (drep2^ex13 and drep2^ex27) were significantly lower than the scores of isogenic w¹¹¹⁸ controls (Figure 5B,C). Re-expression of drep-2 cDNA by using either the pan-neural driver elav^III-Gal4 or two different KC-specific drivers (30y-Gal4 and mb247-Gal4) was sufficient to rescue STM scores (Figure 5C). Drep-2 is, therefore, required in KCs for normal olfactory STM.

ITM is composed of two memory components, consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM) (Quinn and Dudai, 1976; Tully et al., 1994). ASM and ARM rely on different molecular and neuronal mechanisms (Folkers et al., 1993; Tully et al., 1994; Scheunemann et al., 2012). Amnestic cooling abolishes the labile ASM and is, thus, used to separate both components. The drep-2 mutants exhibited regular ARM but were deficient in ASM (Figure 5D), which was evidenced by memory scores not differing between cooled and untreated drep-2 mutants. This loss of ASM was rescued by re-expression of drep-2 using elav^III-Gal4 or KC-specific mb247-Gal4. We conclude that Drep-2 is required in KCs for both STM and ASM, but not for ARM.

Concurrently, morphologies of drep-2-mutant MBs and PN-KC synapses appeared normal (Figure 5—figure supplement 1). Furthermore, the number of synapses in the MB calyx did not differ between either genotype (Figure 5—figure supplement 1D). Thus, drep-2-mutant phenotypes should not be caused by gross structural developmental aberrations. Instead, given the synaptic localization of the protein, it appeared likely that Drep-2 might intersect with signaling pathways involved in synaptic plasticity.

Functional overlap between Drep-2 and mGluR in olfactory conditioning

PN-KC synapses, as is the case for many excitatory synapses in the Drosophila CNS, use ACh as the main fast neurotransmitter (Gu and O'Dowd, 2006; Yasuyama et al., 2002). Drep-2 colocalized here with nicotinic ACh receptor subunits (Figure 4). In addition, several types of metabotropic receptor are also expressed in the calyx, including GABA_B, dopamine, octopamine, and metabotropic glutamate receptors (Enell et al., 2007; Devaud et al., 2008; Busch et al., 2009; Mao and Davis, 2009; Kanellopoulos et al., 2012). DmGluRA is the single functional metabotropic glutamate receptor (mGluR) in Drosophila (Parmentier et al., 1996), orthologous to mammalian group II/III mGluRs. DmGluRA and the mGluR-associated protein Homer show a characteristic expression throughout the Drosophila brain, with strong expression in the MB calyx (Ramaekers et al., 2001; Diagana et al., 2002; Hamasaka et al., 2007; Urizar et al., 2007; Devaud et al., 2008; Kahsai et al., 2012; Kanellopoulos et al., 2012).

We examined different receptor types and related proteins for colocalization with Drep-2 (not shown). Drep-2 colocalized with both mGluR and Homer throughout the brain (not shown), with very prominent co-labeling in the MB calyx (Figure 6A). This made mGluR signaling a prime candidate for a pathway interacting with Drep-2 function.

Figure 6

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Recently, mGluRs have been found to be important for olfactory conditioning in flies. In that work, a decrease of mGluR levels provoked by RNA interference improved performance scores of the olfactory learning mutant dfmr1 (Kanellopoulos et al., 2012). The same effect was observed upon administration of the mGluR antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP).

We assayed the STM performance of the mGluR mutant dmGluRA^112b to independently confirm a function of mGluRs in olfactory learning (Bogdanik et al., 2004). Indeed, a significant reduction in learning scores was observed in these mutants (Figure 6B). In addition to the mGluR antagonist MPEP (McBride et al., 2005; Bolduc et al., 2008; Tauber et al., 2011; Kanellopoulos et al., 2012), the agonist 1S,3R-1-amino-1,3-cyclopentanedicarboxylate (ACPD) has also been shown to be effective in flies (Parmentier et al., 1996; Hamasaka et al., 2007). We, therefore, tested the effects of both MPEP and ACPD on olfactory learning scores of drep-2 mutants (Figure 6C). To this end, we raised flies on food containing either of the two components throughout development and adulthood. In agreement with our result for dmGluRA mutants (Figure 6B), the antagonist (MPEP) significantly decreased learning scores of wild-type flies when fed throughout development (Figure 6C). By contrast, MPEP did not further reduce the learning ability of drep-2 mutants. However, feeding the mGluR agonist ACPD during development effectively rescued the drep-2^ex13 phenotype (Figure 6C (+ACPD dev+ad)). At the same time, feeding the agonist to controls did not alter their learning scores. When ACPD was fed only to adult animals after eclosion, it had no discernible effect on the performance of drep-2 mutants (Figure 6C (+ACPD adult)). In summary, artificial activation of mGluR receptors starting during development can compensate for the olfactory learning deficits of drep-2 mutants.

In order to further investigate a potential relationship between Drep-2 and mGluRs, we produced drep-2; dmGluRA double mutants. These double mutants showed learning indices that were very similar to the scores of both single mutants (Figure 6D). As the learning deficits of both mutants did not add up to a stronger impairment in double mutants, it is likely that both proteins converge, at least partially, into a common regulatory pathway.

Drep-2 and FMRP found in common complexes

Finally, we began exploring the molecular basis of the behavioral connections between Drep-2 and both mGluR signaling and FMRP. We first examined whether Drep-2 might regulate the protein levels or localization of either mGluR or Homer. However, mGluR and Homer levels appeared unaltered in drep-2 mutants (not shown). Therefore, it appeared more likely for Drep-2 to intersect with signaling processes downstream of the mere metabotropic glutamate receptor complex. We used quantitative affinity purification in combination with mass spectrometry to learn about Drep-2 in vivo interaction partners (Vermeulen et al., 2008; Paul et al., 2011). Unfortunately, the Drep-2^C-Term antibody did not precipitate the endogenous Drep-2 protein sufficiently to allow for such an analysis. Thus, a Drep-2^GFP fusion protein showing a localization pattern identical to endogenous Drep-2 (Figure 7—figure supplement 1) was expressed pan-neurally using elav^c155-Gal4. We isolated protein complexes of this pan-neurally overexpressed Drep-2^GFP from fly heads using anti-GFP beads (Figure 7). Parallel pulldowns, using either plain beads or Drep-2^GFP-negative lysates, were performed as controls for nonspecific binding (Figure 7A). All three pulldowns were conducted in triplicate and processed and analyzed by high-resolution shotgun proteomics. Proteins were quantified by label-free quantification and specific interaction partners were extracted using t-test statistics (Hubner et al., 2010).

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On top of the bait proteins Drep-2 and GFP (the latter because the fusion protein Drep-2^GFP was expressed), 35 proteins were robustly enriched over both controls at a false-positive discovery rate (FDR) of 1% (Figure 7B, Supplementary file 1). In order to visualize which of these 35 core proteins are part of a larger grid of putative interactors, an extended protein network was generated (Figure 7D). Proteins that DroID (Murali et al., 2011) or Flybase (McQuilton et al., 2012) lists as (putative) interactors of any of the 35 core proteins (FDR 1%) were added if they fitted the following three conditions: (i) enriched in the pulldowns (elav^c155; uas-drep-2^GFP flies, GFP beads vs plain beads) at an FDR of 10%, (ii) not enriched in the control experiment (GFP beads, wild-type flies vs elav^c155; uas-drep-2^GFP flies) at an FDR of 10% (to eliminate false-positives), and (iii) a (predicted) interaction with at least two of the 35 core proteins (Supplementary file 2).

Among the proteins found to be significantly enriched were Drep-2, GFP, and Drep-3, a cognate binding partner of Drep-2 (Inohara and Nuñez, 1999; Park and Park, 2012). Thus, we were successful in precipitating proteins interacting with Drep-2 and not merely peptides binding unspecifically. 14 of the 35 putative core interacting proteins are associated with membranes (Figure 7C, Supplementary file 1), consistent with our observation that Drep-2 localizes to the postsynaptic plasma membrane. However, neither DmGluRA nor Homer could be identified in the preparation.

Both mGluR and FMRP regulate local synaptic translation (Bhakar et al., 2012). Interestingly, 10 of the 35 proteins have been implicated in the control of mRNA translation and stability. Network analysis of putative interacting proteins (Figure 7D) underlined a strong connection of Drep-2 with RNA-associated proteins as well. Among them, several proteins regulating translation were found: for example, the eIF4G-related, cap-independent translation initiation factor NAT1/p97/DAP5 (Levy-Strumpf et al., 1997; Hundsdoerfer et al., 2005) and Caprin, a dendritic translational repressor (Shiina et al., 2005). Notably, Argonaute-2, involved in RNA interference (Ketting, 2011), was also among the most highly enriched proteins (Supplementary files 1 and 2). It is interesting in this context that both Caprin and Argonaute-2 bind to FMRP (Ishizuka et al., 2002; Papoulas et al., 2010). Finally, FMRP was identified within Drep-2 complexes at an FDR cutoff of 5% (Supplementary file 2). In fact, we could directly confirm the presence of FMRP in Drep-2^GFP immunoprecipitates by immunoblotting (Figure 7E). FMRP, by contrast, could not be detected in an identically treated control experiment conducted in parallel, in which we precipitated the presynaptic protein Syd-1^GFP (Owald et al., 2010) (Figure 7E). Thus, FMRP appears to be present specifically within Drep-2^GFP complexes.

Since Drep-2 could be detected in protein complexes containing FMRP, translational control processes constitute a possible place of action of Drep-2. However, further mechanistic analysis will have to work out the details of the regulatory functions executed by the novel synaptic protein Drep-2.

We here identify the CIDE-N family protein Drep-2 as a novel synaptic protein expressed in the Drosophila CNS (Figure 2) and important for learning and memory (Figure 5). Loss of Drep-2 did not cause transmission deficits at neuromuscular junctions and photoreceptor synapses (not shown) and we did not observe any structural deficits at synapses (Figure 5—figure supplement 1). Thus, Drep-2 is most likely less important for maintaining either base-line transmission or fundamental synaptic architecture. Instead, it might be involved in the regulation of synaptic signaling and plasticity.

Expression of Drep-2 is particularly strong at postsynaptic densities (PSDs) of synapses between projection neurons (PNs) and Kenyon cells (KCs) in the mushroom body (MB) calyx (Figure 4). At these synapses, acetylcholine is released from PNs upon transmission of odor signals (Busto et al., 2010; Gu and O'Dowd, 2006). While elimination of Drep-2 in the whole animal severely impaired olfactory short-term and anesthesia-sensitive intermediate-term memory (Figure 5), re-expression of Drep-2 restricted to KCs was sufficient to fully rescue these learning deficits. As sensation of electric shock, the unconditioned stimulus in aversive olfactory learning, is mediated via dopamine in the MB lobes (Aso et al., 2012), Drep-2 most likely plays its role at PN::KC synapses during reception of the odor, the conditioned stimulus.

Several types of metabotropic receptors are expressed in the calyx: GABA_B, dopamine, octopamine, and metabotropic glutamate receptors (Enell et al., 2007; Devaud et al., 2008; Busch et al., 2009; Mao and Davis, 2009; Kanellopoulos et al., 2012). Which exact role this metabotropic signaling plays in synaptic plasticity processes at PN::KC synapses is essentially unknown. Motivated by the close match between Drep-2 and DmGluRA localization on the level of individual PSDs of the PN-KC synapse (Figure 6A), we started to address a potential functional connection between Drep-2 and DmGluRA-dependent signaling and behavior. We observed that the learning deficits of dmGluRA and drep-2 single mutants did not add up to a stronger phenotype in double mutants (Figure 6D). Moreover, the drep-2 memory deficit was effectively rescued by pharmacological stimulation of DmGluRA (Figure 6C). Drep-2 might, therefore, affect plasticity processes downstream of DmGluRA. In this pathway, the protein could be required for extracting relevant aspects of the olfactory information (odor discrimination vs generalization) and thereby influence subsequent learning. Interestingly, preliminary experiments indicate that the learning deficits of drep-2 mutants might involve decreased Ca²⁺ responses in KCs (not shown).

How might Drep-2 interfere with metabotropic signaling? We were unable to biochemically detect DmGluRA in complexes containing Drep-2, making a direct interaction between both proteins unlikely. Neither could we find any indication of an influence of Drep-2 on mGluR protein levels or localization (not shown). However, we did confirm the presence of the fragile X mental retardation protein FMRP in complexes containing Drep-2 (Figure 7E). In addition to FMRP, several other additional RNA-associated proteins and translational regulators were found by quantitative affinity purification experiments of Drep-2^GFP complexes, followed by mass spectrometry-based protein identification and quantification (Figure 7C,D, Supplementary files 1 and 2). It is, therefore, a probable scenario that Drep-2 indirectly regulates local synaptic protein synthesis.

Several studies have demonstrated that FMRP antagonizes mGluR-mediated synaptic translation (Bhakar et al., 2012). Notably, we found evidence of a functional antagonism between Drep-2 and FMRP-mediated plasticity: both drep-2 and heterozygous dfmr1 single mutants were deficient in olfactory conditioning, but drep-2^ex13/drep-2^ex13; dfmr1^B55/+ double mutants showed normal performance (Figure 6E). Thus, Drep-2 might be required downstream of mGluR signaling, probably in the context of synaptic translation, counteracting translational repression executed by FMRP. We observed that only chronic activation of mGluRs starting during development could improve the learning scores of drep-2 mutants (Figure 6C). This result could be explained by chronic misregulation of neuronal translation in the mutants, rendering the synapses insensitive to enhanced mGluR signaling later in life.

At first glance, it might appear surprising that drep-2^ex13/drep-2^ex13 and dfmr1^B55/+ mutants cancel each other out. However, evidence for a tightly balanced control over synaptic translation has been provided: mutations in the gene tsc2 cause tuberous sclerosis, a disease phenotypically similar to fragile X syndrome (FXS). Impaired long-term depression (LTD) in tsc2-mutant mice could be rescued by the application of mGluR agonists (Auerbach et al., 2011). Moreover, murine fmr1 mutants showed exaggerated LTD, while the double mutant exhibited normal LTD. This demonstrated that synaptic proteins, if misregulated by either impaired or excessive mGluR-induced translation, impede appropriate LTD. In this manner, misregulation of opposing effectors can cause similar phenotypes, as is the case for the olfactory learning performance of drep-2, dmGluRA, and dfmr1 mutants.

Fragile X associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder occurring in carriers of fragile X premutation repeats which is distinct from FXS. The presence of fragile X rCGG premutation repeats in flies activates the microRNA miR-277, which causes neurodegeneration (Tan et al., 2012). One of the targets negatively regulated by miR-277 is Drep-2. Notably, a putative drep-2 hypomorph was shown to enhance the FXTAS neurodegenerative phenotype. While Drep-2 function per se was not investigated in this study and the mechanistic details remain rather elusive, this independent connection between Drep-2 and a scenario related to FXS is also suggestive.

Additional experiments will be required to both examine whether Drep-2 plays a role during translational regulation and to further explore its relationship to metabotropic signaling. Nevertheless, based on our findings, a function of Drep-2 in regulating mGluR-mediated local translation is a possible scenario, which is now open for further investigation. In mammals, Dff-related CIDE proteins have, up to now, mainly been studied in fat tissue (Yonezawa et al., 2011). However, CIDEc is highly expressed in the mammalian brain (Li et al., 2009). It is now an intriguing possibility that Dff family proteins might play non-apoptotic neuronal and synaptic roles in mammals as well.

1. 1. Ahmed S
   2. Holt M
   3. Riedel D
   4. Jahn R

   (2013) Small-scale isolation of synaptic vesicles from mammalian brain

   Nature Protocols 8:998–1009.

   https://doi.org/10.1038/nprot.2013.053

   • Google Scholar
2. 1. Aso Y
   2. Herb A
   3. Ogueta M
   4. Siwanowicz I
   5. Templier T
   6. Friedrich AB
   7. Ito K
   8. Scholz H
   9. Tanimoto H

   (2012) Three dopamine pathways induce aversive odor memories with different stability

   PLOS Genetics 8:e1002768.

   https://doi.org/10.1371/journal.pgen.1002768

   • Google Scholar
3. 1. Auerbach BD
   2. Osterweil EK
   3. Bear MF

   (2011) Mutations causing syndromic autism define an axis of synaptic pathophysiology

   Nature 480:63–68.

   https://doi.org/10.1038/nature10658

   • Google Scholar
4. 1. Bhakar AL
   2. Dölen G
   3. Bear MF

   (2012) The pathophysiology of fragile X (and what it teaches us about synapses)

   Annual Review of Neuroscience 35:417–443.

   https://doi.org/10.1146/annurev-neuro-060909-153138

   • Google Scholar
5. 1. Bogdanik L
   2. Mohrmann R
   3. Ramaekers A
   4. Bockaert J
   5. Grau Y
   6. Broadie KS
   7. Parmentier ML

   (2004) The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology

   The Journal of Neuroscience 24:9105–9116.

   https://doi.org/10.1523/JNEUROSCI.2724-04.2004

   • Google Scholar
6. 1. Bolduc FV
   2. Bell K
   3. Cox H
   4. Broadie KS
   5. Tully T

   (2008) Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory

   Nature Neuroscience 11:1143–1145.

   https://doi.org/10.1038/nn.2175

   • Google Scholar
7. 1. Busch S
   2. Selcho M
   3. Ito K
   4. Tanimoto H

   (2009) A map of octopaminergic neurons in the Drosophila brain

   The Journal of Comparative Neurology 513:643–667.

   https://doi.org/10.1002/cne.21966

   • Google Scholar
8. 1. Busto GU
   2. Cervantes-Sandoval I
   3. Davis RL

   (2010) Olfactory learning in Drosophila

   Physiology 25:338–346.

   https://doi.org/10.1152/physiol.00026.2010

   • Google Scholar
9. 1. Christiansen F
   2. Zube C
   3. Andlauer TFM
   4. Wichmann C
   5. Fouquet W
   6. Owald D
   7. Mertel S
   8. Leiss F
   9. Tavosanis G
   10. Farca Luna AJ
   11. Luna AJ

   (2011) Presynapses in Kenyon cell Dendrites in the mushroom body calyx of Drosophila

   The Journal of Neuroscience 31:9696–9707.

   https://doi.org/10.1523/JNEUROSCI.6542-10.2011

   • Google Scholar
10. 1. Cox J
    2. Neuhauser N
    3. Michalski A
    4. Scheltema RA
    5. Olsen JV
    6. Mann M

    (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment

    Journal of Proteome Research 10:1794–1805.

    https://doi.org/10.1021/pr101065j

    • Google Scholar
11. 1. Davis RL

    (2004) Olfactory learning

    Neuron 44:31–48.

    https://doi.org/10.1016/j.neuron.2004.09.008

    • Google Scholar
12. 1. Davis RL

    (2011) Traces of Drosophila memory

    Neuron 70:8–19.

    https://doi.org/10.1016/j.neuron.2011.03.012

    • Google Scholar
13. 1. Depner H
    2. Lützkendorf J
    3. Babikir HA
    4. Sigrist SJ
    5. Holt MG

    (2014) Differential centrifugation-based biochemical fractionation of the Drosophila adult CNS

    Nature Protocols.

    https://doi.org/10.1038/nprot.2014.192

    • Google Scholar
14. 1. Devaud JM
    2. Clouet-Redt C
    3. Bockaert J
    4. Grau Y
    5. Parmentier ML

    (2008) Widespread brain distribution of the Drosophila metabotropic glutamate receptor

    Neuroreport 19:367–371.

    https://doi.org/10.1097/WNR.0b013e3282f524c7

    • Google Scholar
15. 1. Diagana TT
    2. Thomas U
    3. Prokopenko SN
    4. Xiao B
    5. Worley PF
    6. Thomas JB

    (2002)

    Mutation of Drosophila homer disrupts control of locomotor activity and behavioral plasticity

    The Journal of Neuroscience 22:428–436.

    • Google Scholar
16. 1. Dubnau J
    2. Chiang AS

    (2013) Systems memory consolidation in Drosophila

    Current Opinion in Neurobiology 23:84–91.

    https://doi.org/10.1016/j.conb.2012.09.006

    • Google Scholar
17. 1. Enari M
    2. Sakahira H
    3. Yokoyama H
    4. Okawa K
    5. Iwamatsu A
    6. Nagata S

    (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.

    Nature 391:43–50.

    https://doi.org/10.1038/34112

    • Google Scholar
18. 1. Enell LE
    2. Hamasaka Y
    3. Kolodziejczyk A
    4. Nässel DR

    (2007) gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABA(B) receptors in relation to the GABA(A) receptor subunit RDL and a vesicular GABA transporter

    The Journal of Comparative Neurology 505:18–31.

    https://doi.org/10.1002/cne.21472

    • Google Scholar
19. 1. Fayyazuddin A
    2. Zaheer MA
    3. Hiesinger PR
    4. Bellen HJ

    (2006) The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila

    PLOS Biology 4:e63.

    https://doi.org/10.1371/journal.pbio.0040063

    • Google Scholar
20. 1. Folkers E
    2. Drain P
    3. Quinn WG

    (1993) Radish, a Drosophila mutant deficient in consolidated memory

    Proceedings of the National Academy of Sciences of USA 90:8123–8127.

    https://doi.org/10.1073/pnas.90.17.8123

    • Google Scholar
21. 1. Graveley BR
    2. Brooks AN
    3. Carlson JW
    4. Duff MO
    5. Landolin JM
    6. Yang L
    7. Artieri CG
    8. van Baren MJ
    9. Boley N
    10. Booth BW
    11. Brown JB
    12. Cherbas L
    13. Davis CA
    14. Dobin A
    15. Li R
    16. Lin W
    17. Malone JH
    18. Mattiuzzo NR
    19. Miller D
    20. Sturgill D
    21. Tuch BB
    22. Zaleski C
    23. Zhang D
    24. Blanchette M
    25. Dudoit S
    26. Eads B
    27. Green RE
    28. Hammonds A
    29. Jiang L
    30. Kapranov P
    31. Langton L
    32. Perrimon N
    33. Sandler JE
    34. Wan KH
    35. Willingham A
    36. Zhang Y
    37. Zou Y
    38. Andrews J
    39. Bickel PJ
    40. Brenner SE
    41. Brent MR
    42. Cherbas P
    43. Gingeras TR
    44. Hoskins RA
    45. Kaufman TC
    46. Oliver B
    47. Celniker SE

    (2011) The developmental transcriptome of Drosophila melanogaster

    Nature 471:473–479.

    https://doi.org/10.1038/nature09715

    • Google Scholar
22. 1. Gross C
    2. Berry-Kravis EM
    3. Bassell GJ

    (2012) Therapeutic strategies in fragile X syndrome: dysregulated mGluR signaling and beyond

    Neuropsychopharmacology 37:178–195.

    https://doi.org/10.1038/npp.2011.137

    • Google Scholar
23. 1. Gu H
    2. O'Dowd DK

    (2006) Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ

    The Journal of Neuroscience 26:265–272.

    https://doi.org/10.1523/JNEUROSCI.4109-05.2006

    • Google Scholar
24. 1. Guo A
    2. Li L
    3. Xia SZ
    4. Feng CH
    5. Wolf R
    6. Heisenberg M

    (1996) Conditioned visual flight orientation in Drosophila: dependence on age, practice, and diet

    Learning & Memory 3:49–59.

    https://doi.org/10.1101/lm.3.1.49

    • Google Scholar
25. 1. Hamasaka Y
    2. Rieger D
    3. Parmentier ML
    4. Grau Y
    5. Helfrich-Förster C
    6. Nässel DR

    (2007) Glutamate and its metabotropic receptor in Drosophilaclock neuron circuits

    The Journal of Comparative Neurology 505:32–45.

    https://doi.org/10.1002/cne.21471

    • Google Scholar
26. 1. Hazelrigg T
    2. Levis R
    3. Rubin GM

    (1984) Transformation of white locus DNA in drosophila: dosage compensation, zeste interaction, and position effects

    Cell 36:469–481.

    https://doi.org/10.1016/0092-8674(84)90240-X

    • Google Scholar
27. 1. Hubner NC
    2. Bird AW
    3. Cox J
    4. Splettstoesser B
    5. Bandilla P
    6. Poser I
    7. Hyman A
    8. Mann M

    (2010) Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

    The Journal of Cell Biology 189:739–754.

    https://doi.org/10.1083/jcb.200911091

    • Google Scholar
28. 1. Hundsdoerfer P
    2. Thoma C
    3. Hentze MW

    (2005) Eukaryotic translation initiation factor 4GI and p97 promote cellular internal ribosome entry sequence-driven translation

    Proceedings of the National Academy of Sciences of USA 102:13421–13426.

    https://doi.org/10.1073/pnas.0506536102

    • Google Scholar
29. 1. Huttner WB
    2. Schiebler W
    3. Greengard P
    4. De Camilli P

    (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation

    The Journal of Cell Biology 96:1374–1388.

    https://doi.org/10.1083/jcb.96.5.1374

    • Google Scholar
30. 1. Inohara N
    2. Nuñez G

    (1999) Genes with homology to DFF/CIDEs found in Drosophila melanogaster

    Cell Death and Differentiation 6:823–824.

    https://doi.org/10.1038/sj.cdd.4400570

    • Google Scholar
31. 1. Inoue S
    2. Shimoda M
    3. Nishinokubi I
    4. Siomi MC
    5. Okamura M
    6. Nakamura A
    7. Kobayashi S
    8. Ishida N
    9. Siomi H

    (2002) A role for the Drosophila fragile X-related gene in circadian output

    Current Biology 12:1331–1335.

    https://doi.org/10.1016/S0960-9822(02)01036-9

    • Google Scholar
32. 1. Ishizuka A
    2. Siomi MC
    3. Siomi H

    (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

    Genes & Development 16:2497–2508.

    https://doi.org/10.1101/gad.1022002

    • Google Scholar
33. 1. Kahsai L
    2. Carlsson MA
    3. Winther AM
    4. Nässel DR

    (2012) Distribution of metabotropic receptors of serotonin, dopamine, GABA, glutamate, and short neuropeptide F in the central complex of Drosophila

    Neuroscience 208:11–26.

    https://doi.org/10.1016/j.neuroscience.2012.02.007

    • Google Scholar
34. 1. Kanellopoulos AK
    2. Semelidou O
    3. Kotini AG
    4. Anezaki M
    5. Skoulakis EM

    (2012) Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila

    The Journal of Neuroscience 32:13111–13124.

    https://doi.org/10.1523/JNEUROSCI.1347-12.2012

    • Google Scholar
35. 1. Ketting RF

    (2011) The many faces of RNAi

    Developmental Cell 20:148–161.

    https://doi.org/10.1016/j.devcel.2011.01.012

    • Google Scholar
36. 1. Krashes MJ
    2. Keene AC
    3. Leung B
    4. Armstrong JD
    5. Waddell S

    (2007) Sequential use of mushroom body neuron subsets during drosophila odor memory processing

    Neuron 53:103–115.

    https://doi.org/10.1016/j.neuron.2006.11.021

    • Google Scholar
37. 1. Kremer MC
    2. Christiansen F
    3. Leiss F
    4. Paehler M
    5. Knapek S
    6. Andlauer TFM
    7. Förstner F
    8. Kloppenburg P
    9. Sigrist SJ
    10. Tavosanis G

    (2010) Structural long-term changes at mushroom body input synapses

    Current Biology 20:1938–1944.

    https://doi.org/10.1016/j.cub.2010.09.060

    • Google Scholar
38. 1. Krueger DD
    2. Bear MF

    (2011) Toward fulfilling the promise of molecular medicine in fragile X syndrome

    Annual Review of Medicine 62:411–429.

    https://doi.org/10.1146/annurev-med-061109-134644

    • Google Scholar
39. 1. Laemmli UK

    (1970)

    Cleavage of structural proteins during the assembly of the head of bacteriophage T4

    Nature 227:680–685.

    • Google Scholar
40. 1. Leiss F
    2. Groh C
    3. Butcher NJ
    4. Meinertzhagen IA
    5. Tavosanis G

    (2009a) Synaptic organization in the adult Drosophila mushroom body calyx

    The Journal of Comparative Neurology 517:808–824.

    https://doi.org/10.1002/cne.22184

    • Google Scholar
41. 1. Leiss F
    2. Koper E
    3. Hein I
    4. Fouquet W
    5. Lindner J
    6. Sigrist S
    7. Tavosanis G

    (2009b) Characterization of dendritic spines in the Drosophila central nervous system

    Developmental Neurobiology 69:221–234.

    https://doi.org/10.1002/dneu.20699

    • Google Scholar
42. 1. Levy-Strumpf N
    2. Deiss LP
    3. Berissi H
    4. Kimchi A

    (1997)

    DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death

    Molecular and Cellular Biology 17:1615–1625.

    • Google Scholar
43. 1. Li YH
    2. Lei T
    3. Chen XD
    4. Xia T
    5. Peng Y
    6. Long QQ
    7. Zhang J
    8. Feng SQ
    9. Zhou L
    10. Yang ZQ

    (2009) Molecular cloning, chromosomal location and expression pattern of porcine CIDEa and CIDEc

    Molecular Biology Reports 36:575–582.

    https://doi.org/10.1007/s11033-008-9216-5

    • Google Scholar
44. 1. Lin DM
    2. Goodman CS

    (1994) Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance

    Neuron 13:507–523.

    https://doi.org/10.1016/0896-6273(94)90022-1

    • Google Scholar
45. 1. Luo L
    2. Liao YJ
    3. Jan LY
    4. Jan YN

    (1994) Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion

    Genes & Development 8:1787–1802.

    https://doi.org/10.1101/gad.8.15.1787

    • Google Scholar
46. 1. Mao Z
    2. Davis RL

    (2009) Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity

    Frontiers in Neural Circuits 3:5.

    https://doi.org/10.3389/neuro.04.005.2009

    • Google Scholar
47. 1. McBride SM
    2. Choi CH
    3. Schoenfeld BP
    4. Bell AJ
    5. Liebelt DA
    6. Ferreiro D
    7. Choi RJ
    8. Hinchey P
    9. Kollaros M
    10. Terlizzi AM
    11. Ferrick NJ
    12. Koenigsberg E
    13. Rudominer RL
    14. Sumida A
    15. Chiorean S
    16. Siwicki KK
    17. Nguyen HT
    18. Fortini ME
    19. McDonald TV
    20. Jongens TA

    (2010) Pharmacological and genetic reversal of age-dependent cognitive deficits attributable to decreased presenilin function

    The Journal of Neuroscience 30:9510–9522.

    https://doi.org/10.1523/JNEUROSCI.1017-10.2010

    • Google Scholar
48. 1. McBride SM
    2. Choi CH
    3. Wang Y
    4. Liebelt D
    5. Braunstein E
    6. Ferreiro D
    7. Sehgal A
    8. Siwicki KK
    9. Dockendorff TC
    10. Nguyen HT
    11. McDonald TV
    12. Jongens TA

    (2005) Pharmacological Rescue of synaptic plasticity, Courtship behavior, and mushroom body defects in a Drosophila model of fragile x syndrome

    Neuron 45:753–764.

    https://doi.org/10.1016/j.neuron.2005.01.038

    • Google Scholar
49. 1. McQuilton P
    2. St Pierre SE
    3. Thurmond J
    4. FlyBase Consortium

    (2012) FlyBase 101–the basics of navigating FlyBase

    Nucleic Acids Research 40:D706–D714.

    https://doi.org/10.1093/nar/gkr1030

    • Google Scholar
50. 1. Mukae N
    2. Yokoyama H
    3. Yokokura T
    4. Sakoyama Y
    5. Sakahira H
    6. Nagata S

    (2000) Identification and developmental expression of inhibitor of caspase-activated DNase (ICAD) in Drosophila melanogaster

    The Journal of Biological Chemistry 275:21402–21408.

    https://doi.org/10.1074/jbc.M909611199

    • Google Scholar
51. 1. Murali T
    2. Pacifico S
    3. Yu J
    4. Guest S
    5. Roberts GG III
    6. Finley RL Jr

    (2011) DroID 2011: a comprehensive, integrated resource for protein, transcription factor, RNA and gene interactions for Drosophila

    Nucleic Acids Research 39:D736–D743.

    https://doi.org/10.1093/nar/gkq1092

    • Google Scholar
52. 1. Okamura K
    2. Ishizuka A
    3. Siomi H
    4. Siomi MC

    (2004) Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways

    Genes & Development 18:1655–1666.

    https://doi.org/10.1101/gad.1210204

    • Google Scholar
53. 1. Owald D
    2. Fouquet W
    3. Schmidt M
    4. Wichmann C
    5. Mertel S
    6. Depner H
    7. Christiansen F
    8. Zube C
    9. Quentin C
    10. Körner J
    11. Urlaub H
    12. Mechtler K
    13. Sigrist SJ

    (2010) A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

    The Journal of Cell Biology 188:565–579.

    https://doi.org/10.1083/jcb.200908055

    • Google Scholar
54. 1. Owald D
    2. Khorramshahi O
    3. Gupta VK
    4. Banovic D
    5. Depner H
    6. Fouquet W
    7. Wichmann C
    8. Mertel S
    9. Eimer S
    10. Reynolds E
    11. Holt M
    12. Aberle H
    13. Sigrist SJ

    (2012) Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly

    Nature Neuroscience 15:1219–1226.

    https://doi.org/10.1038/nn.3183

    • Google Scholar
55. 1. Panneels V
    2. Eroglu C
    3. Cronet P
    4. Sinning I

    (2003) Pharmacological characterization and immunoaffinity purification of metabotropic glutamate receptor from Drosophila overexpressed in Sf9 cells

    Protein Expression and Purification 30:275–282.

    https://doi.org/10.1016/S1046-5928(03)00100-1

    • Google Scholar
56. 1. Papoulas O
    2. Monzo KF
    3. Cantin GT
    4. Ruse C
    5. Yates JR
    6. Ryu YH
    7. Sisson JC

    (2010) dFMRP and Caprin, translational regulators of synaptic plasticity, control the cell cycle at the Drosophila mid-blastula transition

    Development 137:4201–4209.

    https://doi.org/10.1242/dev.055046

    • Google Scholar
57. 1. Park OK
    2. Park HH

    (2012) Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3

    FEBS Letters 586:3085–3089.

    https://doi.org/10.1016/j.febslet.2012.07.056

    • Google Scholar
58. 1. Park OK
    2. Park HH

    (2013) A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4

    Apoptosis 18:385–392.

    https://doi.org/10.1007/s10495-013-0815-9

    • Google Scholar
59. 1. Parks AL
    2. Cook KR
    3. Belvin M
    4. Dompe NA
    5. Fawcett R
    6. Huppert K
    7. Tan LR
    8. Winter CG
    9. Bogart KP
    10. Deal JE
    11. Deal-Herr ME
    12. Grant D
    13. Marcinko M
    14. Miyazaki WY
    15. Robertson S
    16. Shaw KJ
    17. Tabios M
    18. Vysotskaia V
    19. Zhao L
    20. Andrade RS
    21. Edgar KA
    22. Howie E
    23. Killpack K
    24. Milash B
    25. Norton A
    26. Thao D
    27. Whittaker K
    28. Winner MA
    29. Friedman L
    30. Margolis J
    31. Singer MA
    32. Kopczynski C
    33. Curtis D
    34. Kaufman TC
    35. Plowman GD
    36. Duyk G
    37. Francis-Lang HL

    (2004) Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome

    Nature Genetics 36:288–292.

    https://doi.org/10.1038/ng1312

    • Google Scholar
60. 1. Parmentier ML
    2. Pin JP
    3. Bockaert J
    4. Grau Y

    (1996)

    Cloning and functional expression of a Drosophila metabotropic glutamate receptor expressed in the embryonic CNS

    The Journal of Neuroscience 16:6687–6694.

    • Google Scholar
61. 1. Parnas D
    2. Haghighi AP
    3. Fetter RD
    4. Kim SW
    5. Goodman CS

    (2001) Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix

    Neuron 32:415–424.

    https://doi.org/10.1016/S0896-6273(01)00485-8

    • Google Scholar
62. 1. Paul FE
    2. Hosp F
    3. Selbach M

    (2011) Analyzing protein-protein interactions by quantitative mass spectrometry

    Methods 54:387–395.

    https://doi.org/10.1016/j.ymeth.2011.03.001

    • Google Scholar
63. 1. Quinn WG
    2. Dudai Y

    (1976) Memory phases in Drosophila

    Nature 262:576–577.

    https://doi.org/10.1038/262576a0

    • Google Scholar
64. 1. Ramaekers A
    2. Parmentier ML
    3. Lasnier C
    4. Bockaert J
    5. Grau Y

    (2001) Distribution of metabotropic glutamate receptor DmGlu-A in Drosophila melanogaster central nervous system

    The Journal of Comparative Neurology 438:213–225.

    https://doi.org/10.1002/cne.1310

    • Google Scholar
65. 1. Rappsilber J
    2. Ishihama Y
    3. Mann M

    (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics

    Analytical Chemistry 75:663–670.

    https://doi.org/10.1021/ac026117i

    • Google Scholar
66. 1. Schägger H

    (2006) Tricine-SDS-PAGE

    Nature Protocols 1:16–22.

    https://doi.org/10.1038/nprot.2006.4

    • Google Scholar
67. 1. Scheunemann L
    2. Jost E
    3. Richlitzki A
    4. Day JP
    5. Sebastian S
    6. Thum AS
    7. Efetova M
    8. Davies S-A
    9. Schwärzel M

    (2012) Consolidated and labile odor memory are separately Encoded within the Drosophila brain

    The Journal of Neuroscience 32:17163–17171.

    https://doi.org/10.1523/JNEUROSCI.3286-12.2012

    • Google Scholar
68. 1. Schwaerzel M
    2. Heisenberg M
    3. Zars T

    (2002) Extinction antagonizes olfactory memory at the subcellular level

    Neuron 35:951–960.

    https://doi.org/10.1016/S0896-6273(02)00832-2

    • Google Scholar
69. 1. Shiina N
    2. Shinkura K
    3. Tokunaga M

    (2005) A novel RNA-binding protein in neuronal RNA granules: regulatory machinery for local translation

    The Journal of Neuroscience 25:4420–4434.

    https://doi.org/10.1523/JNEUROSCI.0382-05.2005

    • Google Scholar
70. 1. Sigrist SJ
    2. Reiff DF
    3. Thiel PR
    4. Steinert JR
    5. Schuster CM

    (2003)

    Experience-dependent strengthening of Drosophila neuromuscular junctions

    The Journal of Neuroscience 23:6546–6556.

    • Google Scholar
71. 1. Song Z
    2. Guan B
    3. Bergman A
    4. Nicholson DW
    5. Thornberry NA
    6. Peterson EP
    7. Steller H

    (2000) Biochemical and genetic interactions between Drosophila caspases and the proapoptotic genes rpr, hid, and grim

    Molecular and Cellular Biology 20:2907–2914.

    https://doi.org/10.1128/MCB.20.8.2907-2914.2000

    • Google Scholar
72. 1. Stocker RF
    2. Heimbeck G
    3. Gendre N
    4. de Belle JS

    (1997) Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons

    Journal of Neurobiology 32:443–456.

    https://doi.org/10.1002/(SICI)1097-4695(199705)32:53.0.CO;2-5

    • Google Scholar
73. 1. Tan H
    2. Poidevin M
    3. Li H
    4. Chen D
    5. Jin P

    (2012) MicroRNA-277 modulates the neurodegeneration caused by Fragile X premutation rCGG repeats

    PLOS Genetics 8:e1002681.

    https://doi.org/10.1371/journal.pgen.1002681

    • Google Scholar
74. 1. Tauber JM
    2. Vanlandingham PA
    3. Zhang B

    (2011) Elevated levels of the vesicular monoamine transporter and a novel repetitive behavior in the Drosophila model of fragile X syndrome

    PLOS ONE 6:e27100.

    https://doi.org/10.1371/journal.pone.0027100

    • Google Scholar
75. 1. Tully T
    2. Quinn WG

    (1985) Classical conditioning and retention in normal and mutant Drosophila melanogaster

    Journal of Comparative Physiology A, Sensory, Neural, and Behavioral Physiology 157:263–277.

    https://doi.org/10.1007/BF01350033

    • Google Scholar
76. 1. Tully T
    2. Preat T
    3. Boynton S
    4. Del Vecchio M

    (1994) Genetic dissection of consolidated memory in Drosophila

    Cell 79:35–47.

    https://doi.org/10.1016/0092-8674(94)90398-0

    • Google Scholar
77. 1. Tusher VG
    2. Tibshirani R
    3. Chu G

    (2001) Significance analysis of microarrays applied to the ionizing radiation response

    Proceedings of the National Academy of Sciences of USA 98:5116–5121.

    https://doi.org/10.1073/pnas.091062498

    • Google Scholar
78. 1. Urizar NL
    2. Yang Z
    3. Edenberg HJ
    4. Davis RL

    (2007) Drosophila homer is required in a small set of neurons including the ellipsoid body for normal ethanol sensitivity and tolerance

    The Journal of Neuroscience 27:4541–4551.

    https://doi.org/10.1523/JNEUROSCI.0305-07.2007

    • Google Scholar
79. 1. Vermeulen M
    2. Hubner NC
    3. Mann M

    (2008) High confidence determination of specific protein-protein interactions using quantitative mass spectrometry

    Current Opinion in Biotechnology 19:331–337.

    https://doi.org/10.1016/j.copbio.2008.06.001

    • Google Scholar
80. 1. Wagh DA
    2. Rasse TM
    3. Asan E
    4. Hofbauer A
    5. Schwenkert I
    6. Dürrbeck H
    7. Buchner S
    8. Dabauvalle MC
    9. Schmidt M
    10. Qin G
    11. Wichmann C
    12. Kittel R
    13. Sigrist SJ
    14. Buchner E

    (2006) Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila

    Neuron 49:833–844.

    https://doi.org/10.1016/j.neuron.2006.02.008

    • Google Scholar
81. 1. Waites CL
    2. Leal-Ortiz SA
    3. Andlauer TF
    4. Sigrist SJ
    5. Garner CC

    (2011) Piccolo regulates the dynamic assembly of presynaptic f-actin

    The Journal of Neuroscience 31:14250–14263.

    https://doi.org/10.1523/JNEUROSCI.1835-11.2011

    • Google Scholar
82. 1. Wolff T
    2. Ready DF

    (1991)

    Cell death in normal and rough eye mutants of Drosophila

    Development 113:825–839.

    • Google Scholar
83. 1. Wu C
    2. Zhang Y
    3. Sun Z
    4. Li P

    (2008) Molecular evolution of Cide family proteins: novel domain formation in early vertebrates and the subsequent divergence

    BMC Evolutionary Biology 8:159.

    https://doi.org/10.1186/1471-2148-8-159

    • Google Scholar
84. 1. Yang M
    2. Armstrong JD
    3. Vilinsky I
    4. Strausfeld N
    5. Kaiser K

    (1995) Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns

    Neuron 15:45–54.

    https://doi.org/10.1016/0896-6273(95)90063-2

    • Google Scholar
85. 1. Yasuyama K
    2. Salvaterra PM

    (1999) Localization of choline acetyltransferase-expressing neurons in Drosophila nervous system

    Microscopy Research and Technique 45:65–79.

    https://doi.org/10.1002/(SICI)1097-0029(19990415)45:23.0.CO;2-0

    • Google Scholar
86. 1. Yasuyama K
    2. Meinertzhagen IA
    3. Schürmann F-W

    (2002) Synaptic organization of the mushroom body calyx in Drosophila melanogaster

    The Journal of Comparative Neurology 445:211–226.

    https://doi.org/10.1002/cne.10155

    • Google Scholar
87. 1. Yokoyama H
    2. Mukae N
    3. Sakahira H
    4. Okawa K
    5. Iwamatsu A
    6. Nagata S

    (2000) A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster

    The Journal of Biological Chemistry 275:12978–12986.

    https://doi.org/10.1074/jbc.275.17.12978

    • Google Scholar
88. 1. Yonezawa T
    2. Kurata R
    3. Kimura M
    4. Inoko H

    (2011) Which CIDE are you on? Apoptosis and energy metabolism

    Molecular Biosystems 7:91–100.

    https://doi.org/10.1039/c0mb00099j

    • Google Scholar
89. 1. Zars TD
    2. Fischer M
    3. Schulz R
    4. Heisenberg M

    (2000) Localization of a short-term memory in Drosophila

    Science 288:672–675.

    https://doi.org/10.1126/science.288.5466.672

    • Google Scholar

Author details

1. Till F M Andlauer

   1. Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany
   2. Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
   3. Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

   Contribution

   TFMA, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-2917-5889

2. Sabrina Scholz-Kornehl

   Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany

   Contribution

   SSK, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Rui Tian

   1. Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany
   2. Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Julius-Maximilians-Universität Würzburg, Würzburg, Germany

   Contribution

   RT, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Marieluise Kirchner

   Department of Cell Signalling and Mass Spectrometry, Max-Delbrück-Centrum für Molekulare Medizin, Berlin-Buch, Germany

   Contribution

   MK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Husam A Babikir

   Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany

   Contribution

   HAB, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Harald Depner

   Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany

   Contribution

   HD, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Bernhard Loll

   Institute of Chemistry and Biochemisty, Freie Universität Berlin, Berlin, Germany

   Contribution

   BL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

8. Christine Quentin

   Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany

   Contribution

   CQ, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

9. Varun K Gupta

   Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany

   Contribution

   VKG, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

10. Matthew G Holt

    Department Laboratory of Glia Biology, Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, Katholieke Universiteit Leuven, Leuven, Belgium

    Contribution

    MGH, Conception and design, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

11. Shubham Dipt

    Department of Molecular Neurobiology of Behavior, Georg-August-Universität Göttingen, Göttingen, Germany

    Contribution

    SD, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

12. Michael Cressy

    Department of Neuroscience, Cold Spring Harbor Laboratory, Cold Spring Harbor, United States

    Contribution

    MC, Acquisition of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

13. Markus C Wahl

    Institute of Chemistry and Biochemisty, Freie Universität Berlin, Berlin, Germany

    Contribution

    MCW, Conception and design, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

14. André Fiala

    Department of Molecular Neurobiology of Behavior, Georg-August-Universität Göttingen, Göttingen, Germany

    Contribution

    AF, Conception and design, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

15. Matthias Selbach

    Department of Cell Signalling and Mass Spectrometry, Max-Delbrück-Centrum für Molekulare Medizin, Berlin-Buch, Germany

    Contribution

    MSe, Conception and design, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

16. Martin Schwärzel

    Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany

    Contribution

    MSc, Conception and design, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

17. Stephan J Sigrist

    1. Genetics, Institute of Biology, Freie Universität Berlin, Berlin, Germany
    2. NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany

    Contribution

    SJS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    stephan.sigrist@fu-berlin.de

    Competing interests

    The authors declare that no competing interests exist.

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