The carboxypeptidase D homolog silver regulates memory formation via insulin pathway in Drosophila

Protein & Cell, Jul 2016

Binyan Lu, Yi Zhao, Jie Zhao, Xiaoyang Yao, Yichun Shuai, Weiwei Ma, Yi Zhong

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The carboxypeptidase D homolog silver regulates memory formation via insulin pathway in Drosophila

© The Author(s) 2016. This article is published with open access at and The carboxypeptidase D homolog silver regulates memory formation via insulin pathway in Drosophila Dear Editor - What is the molecular basis of memory formation? Many genes have been implicated in this process, including those involved in neural cell adhesion, mRNA transport, translation control, and cAMP-PKA signaling. Drosophila, with easy accessibility to genetic, molecular, and behavioral analyses, was also employed in olfactory learning studies and many key genes underlying memory formation were identified in these studies (Bellen et al., 2010) . The majority of identified genes have been shown to be intensively expressed in the mushroom body (MB) of the Drosophila brain (Davis, 2005), including rut (adenylyl cyclase), DCO (the catalytic subunit of PKA), and AKAP Yu (Heisenberg, 2003; Davis, 2005; Lu et al., 2007) . However, these genes are far from enough to understand memory formation comprehensively. In order to identify more genes involved in memory formation, we previously generated 2,667 enhancer trap mutants, each of which contains a p-element (P{GawB}) insertion (Liu et al., 2008) . The insertion includes a Gal4 sequence that can be used to label the expression pattern of the disrupted gene and 368 mutants were selected because their perturbed-gene expression enriched in the mushroom body. These selected mutants were then screened for 3 h memory performance in a well-defined olfactory conditioning paradigm, and one strain (No. 1021) was found defective. Plasmid rescue showed that the p-element of this mutant located in the first intron of the gene silver (svr) (Fig. 1A). Western blot analysis revealed that protein expression level of Svr was significantly decreased in this mutant (svr1021, Fig. 1B). Svr has been reported to involve in viability and behaviors such as cold and ethanol sensitivity, as well as long-term memory in courtship behavior (Sidyelyeva et al., 2010) . To explore the function of Svr in olfactory memory formation, we tested the performance index of svr1021 in different time points after training. Memory of the svr mutant (svr1021) exhibited a significant impairment at both 3 min and 3 h after one-cycle training (Fig. 1C). And 24 h memory of the mutant was also disrupted in svr1021 mutant after spaced training (Fig. 1C). The memory defect was unlikely to be caused by deficiency in sensorimotor system since no abnormality in shock or odor avoidance was observed (Table S1). Finally, in order to confirm that the 3 min memory defect is not a result of random background mutation, we conducted a genetic complementation experiment, using svr1021 and another independent P-element insertion allele, svrKG02090. Indeed, although both heterozygote mutants (svr1021/+ and svrKG02090/+) had normal 3 min memory, such memory was impaired in doubleheterozygote mutant (svr1021/svrKG02090) (Fig. 1D). One previous study showed that svr is involved in Drosophila development (Sidyelyeva et al., 2010) . To figure out whether the disrupted memory formation of silver mutant resulted from the abnormal development or the interference of physiological process of neural system, we acutely manipulated the expression of svr+ transgene in adult flies with the TARGET system. In this system, the Gal4-induced expression is suppressed by a ubiquitously expressed Gal80ts protein at the permissive temperature (18°C), but not at the restrictive temperature (30°C). The gene svr contains three carboxypeptidase domains, and has five endogenous transcriptional forms as a result of alternative splicing (Sidyelyeva et al., 2010) . Previous findings suggested that the functions of CPD domain 1 and 2 are largely redundant, and the inactive CPD domain 3 is required for fully rescuing the mutant’s phenotype (Sidyelyeva et al., 2010) . As a result, we used a longer form containing all three CPD domains (UAS::svr1B-2-3-t1 construct) to restore memory (Sidyelyeva et al., 2010) . Acute expression of svr transgene in svr-labeled-neurons of svr mutant flies (svr1021/Y; UAS-svr/+; Gal80ts/+) effectively rescued the perturbed memory in svr mutant to a comparable level in the control group (UAS-svr/ +; Gal80ts/+) (left panel, Fig. 1E). On the other hand, no significant difference between svr acute expression group (svr1021/Y; UAS-svr/+; Gal80ts/+) and svr mutant group (svr1021/Y) was detected in un-induced conditions (right panel, Fig. 1E). These results suggest that Svr interferes the physiological process of memory formation. Next, in order to find out the functioning neural circuit of Svr in fly brain, we visualized the Gal4 expression pattern of svr1021 using GFP labeling. Confocal imaging of the GFP signal in svr1021/+; UAS-mCD8::GFP/+ revealed a preferential expression in two major compartments: the MB and a small group of Svr Actin * * * * 1 × 3min 1 × 3hr 10 × spaced svr1021/Y * ** UAS-svr/+; GAL80ts/+ svr1021/Y; UAS-svr/+; GAL80ts/+ n.s. ** C E 80 70 neurosecretory cells located in the dorsal/medial region of the fly brain (Fig. 2A and 2B). Other svr enhancer trap lines svrNP2073 and svrNP3600 showed similar expression patterns to svr1021 (Fig. 2A). And the memory formation of these svr-GAL4 mutants was also impaired (Fig. S1). Considering the fact that insulin-producing cells (IPCs) overlap with this cluster of neurosecretory cells ( Nässel et al., 2013 ), we proposed that both the MB and IPC could be candidate brain areas where svr affects memory formation. The GAL4-UAS binary system was used to specifically express svr+ transgene in these cells of interest. We took the OK107-Gal4 to cover all mushroom body neurons, and dilp2-Gal4 to cover the IPCs ( Nässel et al., 2013 ). Subsequent behavioral assays showed that specific expression of svr in the IPCs (svrKG02090/Y; UAS-svr/+; dilp2-Gal4/+) rescued the memory deficiency in svr mutant (svrKG02090/Y; UAS-svr/+, Fig. 2D). However, svr expression in the MB (svrKG02090/Y; UAS-svr/+; OK107-Gal4/+), was not able to generate similar rescuing effect (Fig. 2C). Thus, to our surprise, IPCs rather than MB are the crucial region where Svr influences memory formation. OK107-Gal4/+ svrKG02090/Y; UAS-svr/+ svrKG02090/+; UAS-svr/+; OK107-Gal4/+ dilp2-Gal4/+ svrKG02090/Y; UAS-svr/+ svrKG02090/Y; UAS-svr/+; dilp2-Gal4/+ elav/+ svrKG02090/Y; elav/+ svrKG02090/Y; USA-lnRdel/+; elav/+ * * Drosophila IPCs are functionally similar to mammalian pancreatic islet β cells, because they produce several kinds of Drosophila insulin-like peptides (dilp1-dilp8), which have analogous functio ns to insulin (Nässel et al., 2013 ). Apart from the modulation of energy homeostasis, insulin and its receptors also participate in cognitive processes in the central nervous system (Babri et al., 2007) . Previous studies showed that in both rats and humans, appropriate increase of insulin in specific brain areas can improve certain cognitive abilities, such as spatial memory (Benedict et al., 2004; Babri et al., 2007) . In addition, in C. elegans, insulin/IGF-1 receptor mutant daf-2 has augmented short-term and longterm memory performance in early adulthood (Kauffman et al., 2010). All these findings suggest the participation of insulin pathway in memory formation, although the responsible mechanism has not been clarified. In the present study, Svr functioning in a restricted group of IPCs—dilp2 neurons to regulate memory formation implies that the potential connection between Svr and insulin pathway may be involved in memory formation (Fig. 2D). Silver encodes the homolog of human carboxypeptidase D (CPD) in Drosophila. Carboxypeptidases and endopeptidases can turn precursors into peptides (Fricker, 2005) . CPD is a member of the carboxypeptidase family and has a wide range of substrates, including growth factors, hormones, and neuropeptides (Skidgel and Erdos, 1998) . Another member of the carboxypeptidase family, carboxypeptidase E (CPE) has been reported to cause proinsulin processing defect in its mice mutant (Naggert et al., 1995) . Based on the fact that CPD and CPE share similar enzymatic properties and comparable distribution in the rat central neural system, CPD is speculated to be functionally redundant with CPE (Dong et al., 1999) . Consequently, insulin processing is a potential substrate pathway of svr. The Drosophila insulin/insulin-like growth factor signaling (IIS) system is similar to its human counterpart. It comprises a single insulin receptor (InR) that mediates the function of all eight insulin-like peptides (ILPs), from dilp1 to dilp8 ( Nässel et al., 2013 ). InRs are expressed ubiquitously, but the eight ILPs are expressed in specific tissues, presumably in response to different inputs. Therefore, we overexpressed a constitutively active InR to increase the insulin signal in the pan-neural system of the svr mutant. We found that the expression of constitutively active InR (svrKG02090/Y; UAS-InRdel/+; elav/+) partially rescued the memory impairment in svr mutant (svrKG02090, Fig. 2E). All our findings suggest that Svr regulates the memory formation via insulin pathway in neurosecretory cells outside MB. FOOTNOTES We thank Dr. Lloyd D. Fricker (Albert Einstein College of Medicine), Dr. Ping Shen (University of Georgia), and Bloomington Stock Center for providing stocks. This work was supported by grants from the National Natural Science Foundation of China (Grant No. 91332207, to Y. Z.), the National Basic Research Program (973 Program) ( No. 2013 cb835100, to Y. Z.), the Tsinghua-Peking Joint Center for Life Sciences and the Tsinghua University Initiative Scientific Research Program (20111080956, to Y. Z.). Binyan Lu, Yi Zhao, Jie Zhao, Xiaoyang Yao, Yichun Shuai, Weiwei Ma, and Yi Zhong declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. Binyan Lu1,1,2, Yi Zhao1, Jie Zhao1, Xiaoyang Yao1, Yichun Shuai1, Weiwei Ma1, Yi Zhong1& 1 McGovern Institute for Brain Research, Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China 2 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China & Correspondence: (Y. Zhong) OPEN ACCESS This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Binyan Lu and Yi Zhao have contributed equally to this work. 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Binyan Lu, Yi Zhao, Jie Zhao, Xiaoyang Yao, Yichun Shuai, Weiwei Ma, Yi Zhong. The carboxypeptidase D homolog silver regulates memory formation via insulin pathway in Drosophila, Protein & Cell, 2016, 606-610, DOI: 10.1007/s13238-016-0291-4