The Rap activator Gef26 regulates synaptic growth and neuronal survival via inhibition of BMP signaling

In Drosophila, precise regulation of BMP signaling is essential for normal synaptic growth at the larval neuromuscular junction (NMJ) and neuronal survival in the adult brain. However, the molecular mechanisms underlying fine-tuning of BMP signaling in neurons remain poorly understood. We show that loss of the Drosophila PDZ guanine nucleotide exchange factor Gef26 significantly increases synaptic growth at the NMJ and enhances BMP signaling in motor neurons. We further show that Gef26 functions upstream of Rap1 in motor neurons to restrain synaptic growth. Synaptic overgrowth in gef26 or rap1 mutants requires BMP signaling, indicating that Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling. We also show that Gef26 is involved in the endocytic downregulation of surface expression of the BMP receptors thickveins (Tkv) and wishful thinking (Wit). Finally, we demonstrate that loss of Gef26 also induces progressive brain neurodegeneration through Rap1- and BMP signaling-dependent mechanisms. Taken together, these results suggest that the Gef26-Rap1 signaling pathway regulates both synaptic growth and neuronal survival by controlling BMP signaling.


Introduction
Transsynaptic retrograde signaling from postsynaptic cells controls the development and survival of presynaptic neurons [1][2][3]. At the Drosophila larval neuromuscular junction (NMJ), the bone morphogenetic protein (BMP) ligand glass bottom boat (Gbb) is secreted from the postsynaptic muscle and acts as a key retrograde signal that promotes the expansion of synaptic arbors [4][5][6][7]. In motoneurons, the Gbb signal is processed by a tetrameric presynaptic complex containing the type II BMP receptor wishful thinking (Wit) and either of two type II BMP receptors, thickveins (Tkv) and saxophone (Sax). Upon Gbb binding, this receptor complex phosphorylates the R-Smad mothers against decapentaplegic (Mad). Phosphorylated Mad (P-Mad) translocates into the nucleus through its interaction with the co-Smad Medea to regulate transcription of target genes [8]. Mutations disrupting this canonical BMP signaling pathway, including gbb, wit, tkv, sax, and mad, all display NMJ undergrowth and defective basal transmission [4][5][6][7]. In sharp contrast, genetic conditions to elevate presynaptic BMP signaling cause NMJ overgrowth with excessive formation of small "satellite" boutons [9][10][11][12], which bud off the main axis of the motor axon terminal. Based on these findings, it has been proposed that the level of BMP signaling is instructive for the regulation of NMJ synapse growth [10]. Subsequent work on the Drosophila brain has begun to reveal the importance of precise regulation of BMP signaling in the maintenance of adult neurons. It has been demonstrated that, in addition to synaptic overgrowth, elevation of BMP signaling induces abnormal brain neurodegeneration in the adult fly [9].
Drosophila NMJ studies have identified various endocytic proteins as negative regulators of BMP-dependent synaptic growth. For example, loss of two endocytosis regulators, Dap160/intersectin and endophilin, leads to an increase in synaptic P-Mad levels and NMJ overgrowth with excessive satellite bouton formation [10,13]. In addition, a similar phenotype is also induced by loss of spichthyin (Spict), Spartin, and endosomal maturation defective (Ema), all of which are involved in endolysosomal trafficking of BMP receptors [9,11,14]. Importantly, these endocytic genes are shown to functionally interact with BMP signaling pathway components at the NMJ [9][10][11]14]. These findings imply that endocytosis and subsequent lysosomal degradation of BMP receptors are important mechanisms involved in attenuating Gbb-induced signaling at the NMJ.
In a genetic screen for mutations that affect synaptic morphology at the Drosophila NMJ, we identified the gef26 gene, which encodes a PDZ guanine nucleotide exchange factor (PDZ-GEF) for the small GTPase Rap1. Gef26 was originally known to control the development of various organs primarily by regulating cadherinmediated cell-cell adhesion and integrin-dependent cellmatrix interactions [15][16][17][18][19]. Here, we report a novel role for the Gef26-Rap1 pathway in the regulation of BMPdependent synaptic growth and neuronal survival. Null mutations in the gef26 or rap1 gene cause NMJ overgrowth characterized by excessive satellite bouton formation, recapitulating the phenotype induced by elevated BMP signaling. Genetic interactions between gef26, rap1, and components of the BMP pathway suggest that Gef26 acts through Rap1 to restrain BMPdependent synaptic growth at the NMJ. Importantly, Gef26 promotes endocytic downregulation of surface expression of the BMP receptors Tkv and Wit. Finally, our genetic data indicate that regulation of BMP signaling by the Gef26-Rap1 pathway is critical for neuronal survival in the adult brain.

Results
Drosophila gef26 is required presynaptically for normal synaptic growth To identify genes involved in the regulation of synaptic development, we performed an anatomical screen on 1500 independent EP insertion lines [20,21]. We inspected third instar larval NMJs using the axonal membrane marker anti-HRP. In this screen, we isolated an insertion (G3533) localized in the first intron of the Drosophila gef26 gene (CG9491). These mutants displayed NMJ overgrowth with an excessive formation of small "satellite" boutons (data not shown), which protrude from parental boutons located at primary axon terminal arbors.
To determine the null phenotype of gef26 at the NMJ, we utilized the transheterozygous combination of gef26 6 , a previously reported null allele [19,22], and the Df(2 L)BSC5 deficiency (henceforth referred to as Df ) to delete the gef26 locus. A significant synaptic overgrowth phenotype was observed at every glutamatergic type-I NMJ in gef26 6 /Df third instar larvae. To quantify the gef26 phenotype, we measured overall bouton number and satellite bouton number at NMJ 6/7 and NMJ 4 from abdominal segment 2 (Fig. 1a, b; Additional file 1: Table S1). Compared with wild-type controls (w 1118 ), bouton number normalized to muscle surface area in gef26 6 /Df larvae was increased by 24% at NMJ 6/7 and by 51% at NMJ 4. At the same time, satellite bouton number in gef26 6 /Df was increased by 39% at NMJ 6/7 and by 219% at NMJ 4. Comparable synaptic growth defects were observed in larvae homozygous for gef26 6 ( Fig. 1a, b).
To determine whether gef26 function is required preor postsynaptically for normal synaptic growth regulation, we expressed a gef26 cDNA transgene (UAS-gef26) in gef26 6 /Df mutants under the control of tissue-specific GAL4 drivers. Expression of UAS-gef26 using a neuronal driver (C155-GAL4) fully rescued the NMJ growth defect of gef26 mutants (Fig. 1b). In contrast, expression of UAS-gef26 in all somatic muscles using the BG57-GAL4 driver failed to rescue the NMJ growth defect (Fig. 1b), suggesting that Gef26 functions presynaptically to restrain synaptic growth at the NMJ.
Additional evidence for a presynaptic requirement for Gef26 was provided by assessment of the effect of RNA interference (RNAi)-mediated knockdown of Gef26 expression. Neuronal expression of a dsRNA-fragment of gef26 (UAS-gef26 RNAi ) using C155-GAL4 increased both bouton number and satellite bouton number and mimicked the gef26 loss-of-function mutation, whereas muscular expression of the same dsRNA using BG57-GAL4 had no effect (Additional file 2: Figure S1a, b; Additional file 3: Table S2). This result supports the notion that Gef26 acts in presynaptic neurons to restrain synaptic growth at the NMJ.
We further characterized satellite boutons at gef26 mutant NMJs using several synaptic markers. Satellite boutons contained the active zone antigen NC82 and the synaptic vesicle marker cysteine-string protein (CSP) (Additional file 2: Figure S1c, d). In addition, satellite boutons were found to recruit the subsynaptic reticulum (SSR) marker discs-large (Dlg). Finally, NC82 in satellite boutons was nicely juxtaposed to the essential glutamate receptor subunit GluRIIC (Additional file 2: Figure S1e, f ). Thus, satellite boutons in gef26 mutants display the anatomical hallmarks of functional synapses.
Gef26 acts through Rap1 to regulate synaptic growth Since Gef26 acts via Rap1 to mediate various developmental processes [15-17, 19, 22], we decided to investigate whether Rap1 is the major target for Gef26 in the regulation of synaptic growth. We began by investigating whether loss of rap1 produces NMJ phenotypes similar to those caused by gef26 loss-of-function mutations. For this purpose, we analyzed NMJ morphology in third instar larvae homozygous for the rap1 MI11950 allele (hereafter referred to as rap1 M ) harboring a Minos element within the rap1 gene. Compared with wild-type controls, both overall bouton number and satellite bouton number in rap1 M mutants were significantly increased (Fig. 2a, b; Additional file 4: Table S3). To confirm the requirement for rap1 in the proper regulation of synaptic growth, we also examined NMJ morphology in third instar larvae expressing rap1 dsRNA (UAS-rap1 RNAi ) under the control of C155-GAL4. This genetic manipulation significantly increased overall bouton number and satellite bouton number (Additional file 5: Figure S2a, b; Additional file 6: Table S4). In contrast, muscular expression of UAS-rap1 RNAi did not noticeably alter NMJ morphology (Additional file 5: Figure  S2a, b; Additional file 6: Table S4). Thus, loss of presynaptic rap1 produces gef26-like phenotypes at the NMJ.
Next, we assayed the transheterozygous interaction between gef26 and rap1 during synaptic growth. Heterozygous gef26 6 /+ or rap1 M /+ larvae displayed normal NMJ morphology. However, overall bouton number and satellite bouton number were both significantly increased in transheterozygous gef26 6 /+; rap1 M /+ larvae compared with single gef26 6 / + or rap1 M /+ heterozygotes (Fig. 2b). This type of genetic interaction suggests that Gef26 and Rap1 function in the same pathway.
Finally, we explored the epistatic relationship between gef26 and rap1. Neuronal overexpression of dominantactive Rap1-Q63E (UAS-rap1 CA ) using C155-GAL4 produced an NMJ undergrowth phenotype with fewer synaptic boutons (Fig. 2b). Importantly, neuronal overexpression of UAS-rap1 CA was able to induce a similar phenotype even in the gef26 6 /Df background (Fig. 2b), indicating that the overactivity of Rap1 completely suppresses the synaptic overgrowth in gef26 mutants. These results suggest that Gef26 acts upstream of Rap1 to restrain synaptic growth at the NMJ.

Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling
Previous studies have identified Gbb as a key retrograde signal that stimulates synaptic growth at the NMJ [4][5][6][7]23]. Consistently, elevation of BMP signaling, which can be achieved by either presynaptic overexpression of a dominantly active Tkv receptor or loss of the inhibitory Smad Daughters against decapentaplegic (Dad), causes synaptic overgrowth with excessive satellite bouton formation [9,10], recapitulating phenotypes exhibited by gef26 or rap1 mutants. Therefore, we wondered whether Gef26 and Rap1 might regulate synaptic growth by inhibiting BMP signaling. To test this possibility, we first examined the transheterozygous interaction between gef26 or rap1 and dad at the NMJ. Like gef26 6 /+ and rap1 M /+ larvae, heterozygous dad J1E4 /+ larvae displayed normal NMJ morphology (Fig. 3a, b; Additional file 7: Table S5). In contrast, both overall bouton number and satellite bouton number were significantly increased in transheterozygous gef26 6 /+; dad J1E4 /+ and rap1 M ,+/+,dad J1E4 larvae compared with wild-type controls (Fig. 3a, b), suggesting a functional link between Gef26/Rap1 and the BMP signaling pathway during synaptic growth.
We next examined whether synaptic overgrowth in gef26 or rap1 mutants depends on BMP signaling. Heterozygosity for the BMP receptor gene tkv (tkv 7 /+), which had no effect on NMJ morphology in a wild-type  Table S5). Moreover, removal of both copies of tkv (tkv 1 / tkv 7 ) in the gef26 6 /Df background caused a synaptic undergrowth phenotype, which was similar to that of tkv 1 / tkv 7 mutants (Fig. 3c, d). Thus, BMP signaling is necessary for synaptic overgrowth in gef26 or rap1 mutants.
Finally, we directly tested the role of Gef26/Rap1 in inhibiting BMP signaling by assaying P-Mad levels in gef26 and rap1 mutants. P-Mad accumulation at NMJ synapses and in the nuclei of ventral nerve cord (VNC) motoneurons was significantly increased in gef26 6 /Df or rap1 M /rap1 M larvae compared with wild-type controls (Fig. 3e, f ). Neuronal expression of UAS-gef26 in gef26 6 / Df mutants was capable of reversing the increase of P-Mad in motoneurons (Fig. 3f ), establishing the roles of Gef26 and Rap1 as negative regulators of BMP signaling. These results support a model in which Gef26 and Rap1 restrain synaptic growth by inhibiting BMP signaling.
Gef26 and Rap1 control BMP-dependent synaptic growth by regulating Drosophila fragile X mental retardation 1 (dfmr1) expression and microtubule stability At the Drosophila NMJ, BMP signaling has been shown to repress the expression of the dfmr1 gene [9]. The dfmr1 product (dFMRP) in turn negatively regulates the expression of the microtubule-associated protein 1B (MAP1B) Futsch [24], which promotes synaptic growth by stabilizing synaptic microtubules [25]. Therefore, we hypothesized that Gef26/ Rap1 might control synaptic growth by regulating microtubule stability via the dFMRP-Futsch pathway. To test the involvement of dFMRP in Gef26/Rap1-dependent regulation of synaptic growth, we first examined the transheterozygous interaction between gef26 or rap1 and dfmr1 at the NMJ. Total bouton number and satellite bouton number were significantly higher in transheterozygous gef26 6 /+; dfmr1 Δ50M /+ and rap1 M , +/+,dfmr1 Δ50M larvae than in wild-type controls, although the single heterozygotes displayed normal synaptic growth (Fig. 4a, b; Additional file 8: Table S6). In a subsequent experiment, we directly tested whether loss of Gef26 or Rap1 alters dfmr1 expression. Levels of dfmr1 mRNA were significantly lower in gef26 and rap1 mutants than in wild-type controls, as demonstrated by quantitative realtime PCR (Fig. 4c). Given the roles of Gef26 and Rap1 in inhibiting BMP signaling, these results imply that Gef26/ Rap1 restrains synaptic growth by relieving BMP-dependent repression of dfmr1 transcription.
Futsch reliably labels microtubules in presynaptic motor terminals [25]. Therefore, the above results suggest the involvement of microtubule stability in Gef26/Rap1-mediated regulation of synaptic growth. To directly test this possibility, we assayed the extent of synaptic growth in gef26 and rap1 mutants fed vinblastine, a microtubule-severing drug [26]. When vinblastine was fed at a low concentration (1 μM) that did not affect synaptic growth, it completely suppressed the synaptic overgrowth phenotype of gef26 6 /Df or rap1 M /rap1 M larvae (Fig. 4f, g; Additional file 8: Table  S6). These results support the idea that Gef26/Rap1 controls synaptic growth by regulating microtubule stability via the Futsch pathway.

Gef26 regulates the endocytic internalization of the BMP receptors Tkv and Wit
We next attempted to determine how Gef26 attenuates BMP signaling. Mutations disrupting endocytosis, including endophilin (endo) and dap160, increase presynaptic P-Mad levels at the NMJ along with simultaneous synaptic overgrowth and the formation of excessive satellite boutons [10,13,27], suggesting that endocytosis of surface BMP receptors is an important mechanism to inhibit BMP-dependent synaptic growth. Since a similar phenotype was observed in gef26 mutants, we wondered if Gef26 regulates BMP signaling through endocytosis. To test this possibility, we first investigated genetic interactions between gef26 and mutations in endocytic genes. In heterozygous gef26 6 /+, endoA Δ4 /+, and dap160 Δ1 /+ larvae, total bouton number and satellite bouton number were at wild-type levels ( Fig. 5a, b; Additional file 9: Table S7). In sharp contrast, both parameters were significantly increased in transheterozygous gef26 6 /+; endoA Δ4 /+, or gef26 6 /dap160 Δ1 larvae (Fig.  5a, b), raising the possibility that Gef26 regulates BMPdependent synaptic growth through an endocytic mechanism. It has been proposed that Dap160 interacts with the endosomal protein Nervous wreck (Nwk) to negatively regulate synaptic growth [10,28]. However, total bouton number and satellite bouton number were normal in transheterozygous gef26 6 /+; nwk 2 /+ larvae (Fig. 5b), suggesting that Gef26 and Nwk regulate BMP signaling through distinct pathways.
We then examined the impact of gef26 knockdown on the endocytic internalization of BMP receptors in neuronal BG2-c2 cells. We transiently transfected a Myc-Tkv-Flag or Myc-Wit-Flag construct into control or gef26knockdown cells (Fig. 5c) and prelabeled the cells with an anti-Myc antibody at 4°C. We then initiated endocytosis by incubating the cells at 25°C for 10 min and visualized the internalization of the labeled surface receptors by Myc staining. Total Myc-Tkv-Flag or Myc-Wit-Flag was also monitored by staining for the intracellular Flag-tag after cellular permeabilization. In controls cells, we observed several Myc-Tkv-Flag-or Myc-Wit-Flag-positive intracellular puncta ( Fig. 5d; data not shown). Importantly, when examined in only cells with similar fluorescence intensities of Flag staining, the number of intracellular Myc-Tkv-Flag-or Myc-Wit-Flag-positive puncta per cell was dramatically reduced in gef26-knockdown cells (Fig. 5d, e), suggesting that Gef26 is required for the endocytic internalization of BMP receptors.
Given the role of Gef26 in BMP receptor internalization, we examined whether synaptic vesicle endocytosis is affected in gef26 mutant NMJs. We stimulated third instar fillets with 90 mM K + in the presence of the styryl dye FM1-43FX. During a 1-min labeling period, dye uptake into synaptic boutons was not significantly different between wild-type and gef26 6 /Df mutant animals (Additional file 10: Figure S3a, b). This result indicates that loss of Gef26 does not grossly affect endocytosis at the presynaptic terminal of the NMJ.
Finally, we investigated whether Gef26 collaborates with Rap1 and the BMP pathway to maintain normal locomotor ability and neuronal survival. To this end, we first examined transheterozygous combinations of gef26 and rap1 or dad with respect to locomotor dysfunction. At 20 days of age, transheterozygous gef26 6 /+; rap1 M /+ and gef26 6 /+; dad J1E4 /+ flies displayed mildly reduced climbing response compared with age-matched gef26 6 /+, rap1 M /+, or dad J1E4 /+ flies (data not shown). However, these transheterozygous flies at 30 days of age exhibited severely reduced climbing ability (Additional file 11: Figure S4c, d). We also examined transheterozygous interactions between gef26 and rap1 or dad with respect to brain neurodegeneration. At 20 days of age, heterozygous gef26 6 /+, rap1 M /+, or dad J1E4 /+ flies were not distinguishable from wild-type controls with respect to the total number of vacuoles (Fig. 6f ). In sharp contrast, there was a significant vacuolization in the brains of transheterozygous gef26 6 /+; rap1 M /+ or gef26 6 /+; dad J1E4 /+ flies (Fig. 6f ), supporting a functional link between Gef26, Rap1, and the BMP signaling pathway in the regulation of neuronal survival in the adult brain.
How might Gef26 regulate BMP signaling? Increasing evidence suggests that endocytosis of surface BMP receptors is a key mechanism of signal attenuation at presynaptic NMJ terminals. In support of this notion, our data imply that Gef26 inhibits BMP signaling by regulating the endocytic internalization of its receptor(s). gef26 displays transheterozygous interactions with mutations disrupting endocytosis (i.e., dap160 and endoA) during synaptic growth. In addition, gef26 mutant NMJs show an increase in the level of surface Tkv, supporting the role of Gef26 in receptor endocytosis. Most directly, we show that Gef26 facilitates the endocytic internalization of the BMP receptors Tkv and Wit in cultured cells. These findings imply a model in which Gef26 attenuate BMP signaling through facilitating endocytosis of BMP receptors (Fig. 6g).
Elevated BMP signaling has been implicated in the pathogenesis of hereditary spastic paraplegia (HSP), a group of neurodegenerative motor disorders. In mammalian cells, several HSP proteins, including NIPA1, Spastin, and Spartin, have been shown to inhibit BMP signaling [31]. At the Drosophila NMJ, the NIPA1 homologue Spichthyin (Spict) and Spartin also inhibit BMP signaling to restrain synaptic growth [9,11]. Importantly, it has now been demonstrated that elevation of BMP signaling in adult spartin flies causes progressive neurodegeneration and locomotor dysfunction [9]. Consistent with these studies and the proposed role of Gef26 as an inhibitor of BMP signaling, depletion of gef26 in the adult fly induces neurodegeneration and locomotor function. Thus, the current study solidifies the notion that precise regulation of BMP signaling is critical for the maintenance of adult neurons. A future challenge will be to investigate whether PDZ-GEF1 and other human Gef26 homologues contribute to the maintenance of the human motor system and, if so, whether this neuroprotective role involves the regulation of retrograde BMP transsynaptic signaling.
A final point of interest is the mechanism of how the Gef26-Rap1 pathway facilitates BMP receptor endocytosis. In various experimental systems, Rap1 has been identified to regulate actin-driven cellular processes. For example, mammalian Rap1 promotes cell spreading by localizing the RacGEFs Vav2 and Tiam1 to sites of lamellipodia extension [32], which is driven by Rac-dependent actin polymerization. In addition, Dictyostelium Rap1 is also involved in chemotaxis by activating the Rac signaling pathway through RacGEF1 [33]. Since actin polymerization is known to provide mechanical forces required for multiple stages of endocytosis [34], it is tempting to speculate that Rap1 facilitates endocytosis by regulating actin polymerization through the RacGEF-Rac signaling pathway. Interestingly, the Rac signaling pathway has been implicated in the regulation of BMP-dependent synaptic growth at the Drosophila NMJ [35]. In future studies, it will be interesting to investigate the role of the Rac signaling pathway in Rap1-dependent endocytosis.

Molecular biology
Full-length cDNAs for gef26 and rap1 were obtained by reverse transcription PCR of total RNA extracted from Drosophila S2R+ cells and introduced into the pUAST or pUAST-Myc vector to generate UAS-gef26 and UAS-Myc-rap1. For UAS-Myc-rap1 CA , glutamine 63 was mutated to glutamate by overlapping PCR using UAS-Myc-rap1 (the template DNA) and the primers 5'-ATGGCCGT-GAACTCCTCCGTACCC-3′ and 5'-TACGGAGGAGTT-CACGGCCATGCG-3′ in combination with the BglII-Myc-linked primer 5'-GGGAGATCTGCCACCATG-GAACAAAAACTCATCTCAGAAGAG-GATCT-GATGCGTGAGTACAAAATC-3′ and the XbaI-linked primer 5'-GGGTCTAGATAGCAGAACACATAGGGAC-3′, respectively, and the assembled product was introduced into pUAST. For pAc-Myc-tkv-Flag, a full-length cDNA (clone ID: LD45557) for tkv (CG14026) was obtained from the Drosophila Genomics Resource Center (Bloomington, IN, USA). The cDNA insert was PCRamplified and then introduced into the pTOP Blunt V2 vector (Enzynomics, Daejeon, Republic of Korea). Myc and Flag epitope-tag sequences were introduced immediately downstream of the signal sequence and at the C-terminus of Tkv, respectively, by PCR-based mutagenesis. The resulting Myc-tkv-Flag insert was subcloned into the pAc5.1 vector. For pAc-Myc-wit-Flag, Flag epitope-tag sequence was introduced downstream of the wit sequence of pAc-Myc-wit [9] by PCR-based mutagenesis. The resulting Myc-wit-Flag fragment was re-introduced into the pAc5.1 vector.
To measure levels of dfmr1 expression, total RNA was extracted from the third instar brain and ventral ganglion using the TRIsure kit (Bioline, Taunton, MA, USA) and reverse transcribed using the SuperScript III cDNA synthesis kit (Invitrogen). Quantitative real-time PCR reactions were performed using SYBR Select Master Mix (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7500 Real-Time PCR System. The mean Ct of triplicate reactions was used to determine relative expression of dfmr1 using the 2 -ΔΔCT method. Expression of rp49 was used as the internal control. The primers used were: dfmr1, 5'-GGATCAGAACATAC-CACGTG-3′ and 5'-CTGGCAGCTATCGTGGAGGCG-3′; and rp49, 5'-CACCAGTCGGATCGATATGC-3′ and 5'-CACGTTGTGCACCAGGAACT-3′.
For RNA interference (RNAi) experiments in BG2-c2 cells, gef26 dsRNA was produced by in vitro transcription of a DNA template containing T7 promoter sequences at both ends, as described previously [38]. The DNA template was produced by PCR from the UAS-gef26 vector using primers containing a T7 promotor sequence followed by gef26-specific sequences: 5'-GTGGCCGGCTCTACCAGT-3′ and 5'-TGGTACGC-GAGTCGAACG-3′.

Immunostaining of larval NMJs
Wandering third-instar larvae were dissected in Ca 2 + -free HL3 solution and fixed in PBS containing 4% formaldehyde for 20 min. Fixed larval fillets were washed with PBT-0.1 (PBS, 0.1% Triton X-100) and blocked with PBT-0.1 containing 0.2% BSA for 1 h. Samples were sequentially incubated with primary antibodies overnight at 4°C and fluorescently-labeled secondary antibodies for 1 h at room temperature. The following monoclonal antibodies from the Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA) were used as primary antibodies FITC-and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:200. Images were captured with an LSM 800 laser-scanning confocal microscope using a C Apo 40× W or Plan Apo 63 × 1.4 NA objective.
Quantification of bouton number and satellite bouton number was performed at NMJ 6/7 and NMJ 4 in abdominal segment 2, as previously described [20]. Bouton number was normalized to muscle surface area. Statistical analysis was performed using SigmaPlot (Systat Software, San Jose, CA, USA). Comparisons were made by one-way ANOVA analysis with a post-hoc Turkey test. For comparison of only two samples, an unpaired Student's t-test was used. Data are presented as mean ± SEM.

Histology, immunostaining, and TUNEL staining of adult brains
Heads from adult flies at 2, 10, 20, 30, and 40 days posteclosion were fixed overnight in PBS containing 4% paraformaldehyde at 4°C, embedded in paraffin, and subjected to serial 5-μm sectioning in a frontal orientation. Serial sections covering the entire brain were placed on a single slide and stained with hematoxylin and eosin (H&E) using a standard protocol. Vacuoles larger than 5 μm were counted throughout the entire brain.
For immunostaining analysis, brains from 20-day-old flies were dissected in ice-cold PBS, and fixed overnight in PBS containing 4% formaldehyde at 4°C. Fixed brains were subsequently permeabilized in PBT-0.3 (PBS, 0.3% Triton X-100) for 1 h and blocked with PBT-0.3 containing 5% BSA for 1 h. The brains were sequentially incubated with primary antibodies for 48 h at 4°C and fluorescently-labeled secondary antibodies for 24 h at 4°C. The following primary antibodies were used in this study: anti-Elav (7E8A10, DSHB) at 1:10, anti-Repo (8D12, DSHB) at 1:10, and anti-cleaved caspase-3 (Cell Signaling) at 1:100. Antibody-stained brains were mounted in SlowFade antifade medium (Invitrogen). Fluorescent images were acquired with a LSM 800 laser-scanning confocal microscope using a C Apo 40× W objective.
TUNEL assays on paraffin sections of adult brains were performed using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Briefly, paraffin sections were dewaxed according to standard procedures. After washed with PBS, the sections were permeabilized in PBS containing 0.1% sodium citrate and 0.1% Triton X-100 for 15 min at room temperature. After washing with PBS, the samples were incubated with the TUNEL reaction mixture in a dark humid chamber for 1 h at 37°C, prior to DAPI staining for 5 min at room temperature. TUNEL-and DAPI-positive cells were counted in three consecutive, middle frontal sections of adult brains.

Adult climbing test
Adult locomotor ability was assayed as described previously [9]. For each genotype tested, approximately 100 flies were collected within 1 day of eclosion; aged for 2, 10, 20, 30, and 40 days; and placed into a glass graduated cylinder. After 5 min of adaptation to their environment, flies were gently vortexed for 5 s. The distance climbed by individual flies in a 30 s period was measured. Climbing assays were repeated 3 times for each genotype, and the results were averaged.