Research Papers: Gerotarget (Focus on Aging):

GULP1/CED-6 ameliorates amyloid-β toxicity in a Drosophila model of Alzheimer’s disease

PDF |  HTML  |  How to cite  |  Order a Reprint

Oncotarget. 2017; 8:99274-99283. https://doi.org/10.18632/oncotarget.20062

Metrics: PDF 529 views  |   HTML 1222 views  |   ?  

Wai Yin Vivien Chiu, Alex Chun Koon, Jacky Chi Ki Ngo, Ho Yin Edwin Chan and Kwok-Fai Lau _


Wai Yin Vivien Chiu1,*, Alex Chun Koon1,*, Jacky Chi Ki Ngo1, Ho Yin Edwin Chan1 and Kwok-Fai Lau1

1 School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR

* These authors have contributed equally to this work

Correspondence to:

Kwok-Fai Lau, email:

Keywords: CED-6, APP, Aβ, neurodegeneration, neurotoxicity, Gerotarget

Received: June 13, 2017 Accepted: July 30, 2017 Published: August 08, 2017


Amyloidogenic processing of APP by β- and γ-secretases leads to the generation of amyloid-β peptide (Aβ), and the accumulation of Aβ in senile plaques is a hallmark of Alzheimer’s disease (AD). Understanding the mechanisms of APP processing is therefore paramount. Increasing evidence suggests that APP intracellular domain (AICD) interacting proteins influence APP processing. In this study, we characterized the overexpression of AICDinteractor GULP1 in a Drosophila AD model expressing human BACE and APP695. Transgenic GULP1 significantly lowered the levels of both Aβ1-40 and Aβ1-42 without decreasing the BACE and APP695 levels. Overexpression of GULP1 also reduced APP/BACE-mediated retinal degeneration, rescued motor dysfunction and extended longevity of the flies. Our results indicate that GULP1 regulate APP processing and reduce neurotoxicity in a Drosophila AD model.



Human GULP1 (engulfment adaptor PTB-domain-containing 1), the homologue of Caenorhabditis elegans CED-6, is an adaptor protein with multiple protein interaction domains/regions including an N-terminal phosphotyrosine-binding (PTB) domain, a centrally located leucine zipper and a carboxyl terminal proline/serine rich region [1-3]. GULP1/CED-6 has been implicated in phagocytosis as it interacts with several engulfment receptors including CED-1, stabilin-1 and stabilin-2 [3-6]. Recently, we and others have shown that GULP1 PTB domain binds to Alzheimer’s disease amyloid precursor protein (APP) [7, 8].

APP is a large type I transmembrane protein contains a large ectodomain and a small intracellular domain, namely AICD. Amyloidogenic processing of APP by β- and γ-secretases leads to the generation of amyloid-β peptide (Aβ), and accumulation of Aβ to form senile plaques is a hallmark of Alzheimer’s disease (AD). Although the mechanisms by which APP processing is regulated are still not fully understood, increasing evidence suggests that AICD interacting proteins can influence APP metabolism and Aβ generation (see reviews [9, 10]). In fact, GULP1 is found to alter APP processing and Aβ generation in transfected cells [7, 8]. However, the effect of GULP1 on Aβ production in vivo remains to be determined.

To understand the effect of GULP1 in vivo, we utilized the fruit fly, Drosophila melanogaster. Drosophila is a popular model for studying neurodegenerative diseases, as approximately 75% of all known human disease-associated genes are conserved in flies, including those implicated in AD [11]. In addition, AD phenotypes such as learning disabilities and plaque deposition can also be observed in Drosophila [12, 13]. In this study, we established transgenic flies to overexpress GULP1 in an existing Drosophila AD model which expresses human APP695 wildtype and BACE genes. This Drosophila model is a powerful tool for AD research as the files exhibit a number of clinical AD neuropathology and symptomology for both familial/sporadic AD including Aβ aggregation and memory defects [14]. In this study, we observed that GULP1 reduces Aβ production, reduces retinal degeneration, rescues motor dysfunction and improves the life expectancy of the flies. Our data suggest that GULP1 is a potential modifier of neurotoxicity in AD by lowering Aβ levels.


Characterization of GULP1 transgene expression in Drosophila

To determine the effect of GULP1 in vivo, we created UAS-GULP1 transgenic flies, and crossed them to gmr-GAL4 to overexpress GULP1 in the fly eyes. Expression of GULP1 in gmr-GAL4/+; UAS-GULP1/+ animals (gmr > GULP1) was confirmed by Western blot analysis (data not shown), and there was no significant change in the number of rhabdomeres per ommatidium between gmr > GULP1 heterozygous and control gmr driver flies (Figure 1A). To investigate the effect of GULP1 on AD-like symptoms in flies, we utilized an existing Drosophila AD model which expresses human APP695 and BACE transgenes using gmr-GAL4 (gmr > APP, BACE) [14]. We crossed the UAS-GULP1 flies to the gmr > APP, BACE flies to produce gmr-GAL4/+; UAS-APP695, UAS-BACE/UAS-GULP1 heterozygous flies (gmr > GULP1, APP, BACE). Western blot analysis revealed the expression of GULP1, APP and BACE transgenes in several independent lines (Figure 1B). There was no noticeable difference in GULP1 expression in different lines. The expression of a third transgene (GULP1) in the AD model did not result in a lower expression of APP or BACE due to the dilution of available GAL4. In contrary, the expression level of APP holoprotein was surprisingly increased by approximately 58% in gmr > GULP1, APP, BACE flies as compared to gmr > APP; BACE to flies (Figure 1B).

Characterization of GULP1 transgene in

Figure 1: Characterization of GULP1 transgene in Drosophila. A. Overexpression of GULP1 using gmr-GAL4 does not change the overall retinal structure. Flies were from three independent experiments. The flies were raised at 19 oC and assayed at 5 dpe. B. Overexpression of human APP695, BACE and GULP1 proteins in Drosophila. Immunoblot analysis of fly head homogenates for APP, BACE, GULP1. 1M - 5M were independent transgenic flies of GULP1 under the UAS promoter, which were crossed into the fly disease model of APP and BACE expression driven by gmr-GAL4. Actin was used as loading control. Flies were raised at 28 oC and assayed at 6 dpe.

Overexpression of GULP1 rescues motor dysfunction and extends life span in a Drosophila AD model

Progressive decline in locomotor ability is one of the surrogate markers of neurotoxicity in a number of AD models [14-16]. To investigate if GULP1 can ameliorate APP/BACE-induced motor dysfunction, pan-neuronal elav-GAL4 driver was used to overexpress two independent UAS-GULP1 fly lines (3M and 5M) together with UAS-APP695 and UAS-BACE. Similar to the previous report, we observed that elav > APP, BACE heterozygous flies have reduced climbing activities, indicating that APP and BACE compromised CNS functions [14] (Figure 2A). Remarkably, overexpression of GULP1 significantly improved the APP/BACE-induced motor impairment (Figure 2B).

GULP1 ameliorates APP and BACE-induced locomotor dysfunction in a

Figure 2: GULP1 ameliorates APP and BACE-induced locomotor dysfunction in a Drosophila AD model. A. Compared with control elav-GAL4 driver flies, overexpression of APP and BACE showed significant motor impairment; B. AD models expressing GULP1 delayed the impairment of locomotor function. The average climbing indices of elav driver control, elav > APP, BACE, elav > APP, BACE, GULP1 (3M) and elav > APP, BACE, GULP1 (5M) were 91%, 74%, 90% and 89% respectively. N = 80, *p < 0.05; error bars represent SD from at least 80 flies from three independent experiments. The flies were raised at 19 oC and assayed at 20 dpe.

In addition, consistent with the previous report, the median survival time of elav > APP, BACE flies was markedly shorter than the heterozygous elav-GAL4 control flies (30 days vs 80 days) (Figure 3A and 3D). On the other hand, overexpression of GULP1 increased the lifespan of the AD model to a median survival time to 54 days (Figure 3B-3D).

GULP1 extends life span in a

Figure 3: GULP1 extends life span in a Drosophila AD model. A. Compared with control flies, overexpression of APP and BACE showed significantly shorter life span; B.-C. AD models expressing GULP1 demonstrated significantly longer survival time. D. Quantification of life span. N = 70. Log-rank (Mantel-Cox) test was performed. **** p < 0.0001, ** p = 0.0013, ** p = 0.0039 for A., B. and C. respectively. At least 70 flies from three independent experiments were assayed. The flies were raised at 19 oC.

GULP1 protects against APP/BACE-induced neurodegeneration in a Drosophila AD model

The Drosophila compound eye has been widely used for monitoring neurotoxicity and neurodegeneration [17, 18]. To investigate the hypothesis that GULP1 reduces degeneration in the Drosophila AD model, we examined the ommatidial organization of gmr > GULP1, APP, BACE fly by pseudopupil assay. As shown in Figure 1B, overexpression of GULP1 did not show noticeable effect on the overall Drosophila retinal structure. Yet, GULP1 overexpression reduced the effect of APP/BACE-mediated degeneration as gmr > GULP1, APP, BACE flies showed significant higher average number of rhabdomeres per ommatidium than gmr > APP, BACE flies (Figure 4A-4C).

GULP1 reduces retinal degeneration in a

Figure 4: GULP1 reduces retinal degeneration in a Drosophila AD model. A. Representative photomicrographs of fly retina in AD models with or without GULP1 expression from one of the three independent experiments. Scale bar is 7 µm. B. Distribution pattern of number of visible rhabdomeres per ommatidium in AD models with or without GULP1 expression from one experiment. Similar trend was observed in the other two independent experiments. C. Quantification of visible rhabdomeres. On average, 3.29 rhabdomeres per ommatidium were observed in flies expressing APP695 and BACE. Overexpression of GULP1 significantly suppressed the degeneration induced by APP and BACE expression, and increased the rhabdomere score to an average of 3.95 and 4.43 respectively. N = 300, ***p < 0.01. Flies were from three independent experiments. The flies were raised at 19 oC and assayed at 5 dpe.

Overexpression of GULP1 reduces Aβ generation in a Drosophila AD model

It is known that the overexpression of human APP and BACE increases amyloidogenic processing of APP and generation of Aβ in Drosophila [14, 19], and Aβ has been reported to shorten Drosophila lifespan and cause photoreceptor abnormality in the compound eye [20, 21]. Thus, it is possible that the mitigation of neurotoxicity by GULP1 is due to an overall reduction of Aβ production. To test this hypothesis, we overexpressed GULP1, APP and BACE using gmr-GAL4 and examined GULP1’s effect on Aβ production. Indeed, we observed a significant reduction of Aβ1-40 and Aβ1-42 in gmr > GULP1, APP, BACE flies as compared to gmr > APP, BACE flies (Figure 5A), indicating that GULP1 rescues structural, behavioral and longevity phenotypes in the Drosophila AD model by lowering Aβ production levels.

Active ARF6 has been shown to lower Aβ generation by altering endosomal sorting of BACE1 in various mammalian cell types including primary neurons [22]. Moreover, GULP1 has been shown to alter the generation of APP C-terminal fragment β, a product of BACE1 cleavage of APP, in mammalian cells [7]. It is possible that GULP1 regulates APP processing via ARF6 in some fashion as the PTB domain of GULP1 interacts with ARF6, and the knockdown of GULP1 reduces ARF6 activation in mouse embryo fibroblast MEF 1 [23]. To test the hypothesis, we transfected cells with GULP1, and examined the levels of active ARF6 (ARF6-GTP). While the total ARF6 level did not change, more active ARF6 was observed in cells overexpressing GULP1 (Figure 5B). This suggests that GULP1 possibly reduces Aβ production and neurotoxicity via the activation of ARF6.

Overexpression of GULP1 decreases A&#x3b2;1-40 and A&#x3b2;1-42 levels in a

Figure 5: Overexpression of GULP1 decreases Aβ1-40 and Aβ1-42 levels in a Drosophila AD model. A. Aβ1-40 and Aβ1-42 levels were measured by human Aβ ELISA. Both Aβ1-40 and Aβ1-42 decreased significantly in flies expressing GULP1. **p < 0.01, *p < 0.05; Error bars are SD from at least 120 flies from three independent experiments. The flies were raised at 28 oC and assayed at 6dpe. B. Total ARF6 levels and ARF6-GTP levels were examined in CHO cells overexpressing GULP1 by immunoblotting (Top panel). Bar chart shows relative ARF6-GTP amount. Data were obtained from three independent experiments. N = 3, *p < 0.001. Error bars are SD.


Several lines of evidence from cell models suggest GULP1 modulates APP processing [7, 8]. However, the biological roles of GULP1 on Aβ productions in vivo and the subsequent physiological effects remain unknown. The current study provides first evidence that GULP1 affects human APP metabolism in vivo and improve structural, behavioral and longevity phenotypes in a Drosophila AD model.

The mechanism(s) by which GULP1 alters APP processing is largely unknown. GULP1 has been shown to interact with ARF6 [23], a member of the Ras superfamily of small GTPases functions in trafficking the membrane components between the plasma membrane and endosomal compartments (see review [24]). Several studies have shown that endosomes contain high level of BACE, and are a major subcellular compartment for Aβ production (see review [25]. In fact, active ARF6 has been shown to regulate endosomal sorting of BACE1 and lead to reduction in Aβ generation [22]. Here, we showed that overexpression of GULP1 could induce ARF6 activation. Hence, the effect of GULP1 on APP processing in Drosophila may be via activation of dARF6, the fly homolog of ARF6. Alternatively, dCED-6, the fly homologue of GULP1, has been reported to function as an in vivo clathrin adaptor for clathrin-mediated Yolkless uptake in Drosophila oocytes [26-28]. As APP internalization is also clathrin-mediated [29], it is possible that GULP1 regulates APP endocytosis processing in flies. It is also suggested that GULP1 may reduce maturation of APP along the secretory pathway and impair APP trafficking to the plasma membrane. Such retention of APP proteins in the secretory pathway consequently traps more of them inside the cells, limiting their processing by secretases at the plasma membrane, and ultimately reduced Aβ generation [8]. This is consistent with our observation that the level of APP appeared to be increased when GULP1 was overexpressed (Figure 1B).

In Drosophila, dCED-6 is a key molecule in the Draper pathway mediating the glial engulfment of dying/injured neurons as well as presynaptic debris at the larval neuromuscular junction [26, 30, 31]. Since elav-GAL4’s expression includes both neuronal and glial cells [32], it is possible that the overexpression of GULP1 in glia activated the Draper pathway, and facilitate the engulfment of extracellular Aβ. In fact, many previous studies have demonstrated the role of glial cells in Aβ clearance and degradation [33-38]. Additionally, the suppressive effect of GULP1 on Aβ level and reduced structural and behavioral abnormalities could be a result of potential neuroprotective effect of GULP1 on Aβ clearance as GULP1 is reported to stimulate the signaling of transforming growth factor-β (TGF-β) [39], a neurotrophic cytokine against Aβ toxicity [40].

On the other hand, conflicting effect of GULP1 on APP processing are reported [7, 8]. Similar controversies are reported for other AICD interacting proteins including FE65s and X11s [9, 41-49]. Noteworthy, GULP1, FE65s and X11s are adaptor proteins that functions in recruiting interactors for various biological pathways. One possible reason for such conflicting observations is that the cell types or models employed in the studies expressing different types and amounts of their interactors. Thus, in addition to ARF6, other GULP1 interactors may also participate in regulating APP processing. Moreover, a number of studies have revealed that the phosphorylation status of APP interactors would influence their effects on APP processing [50, 51]. Noteworthy, phosphoproteomic and mass spectrometric analyses from various laboratories have shown that GULP1 is a phosphoprotein [52-55]. Hence, the discrepancy in the effect of GULP1 on APP processing may also be a result of phosphorylation status of GULP1. Therefore, identification of the full spectrum of GULP1 interacting proteins and investigation of the role of GULP1 phosphorylation will provide further mechanistic insights into how GULP1 modulates APP processing.

Although lowering Aβ by the enhancement of Aβ clearance using recombinant Aβ antibodies have shown promises in mouse model [56], Eli Lilly recently announced that their Aβ antibody drug Solanezumab failed to demonstrate efficacy in an 18-month phase III clinical trial with over 2,100 participants. The exact reasons for the failure of the trial remain to be determined [57]. Solanezumab is thought to be function by sequestering Aβ to promote Aβ clearance. However, the drug could not lower Aβ generation. Therefore, combination therapy that increases Aβ clearance and suppresses Aβ production may be an alternative approach for AD. However, current strategies for reducing Aβ production remain unsatisfactory such as the use of γ-secretase inhibitors. Our finding that GULP1 reduces Aβ production in an AD Drosophila model does not only improve our understanding of the function of GULP1 in APP processing in vivo, but also opens a novel avenue for investigating the possibility of targeting GULP1-APP interaction to alter Aβ production.


Generation of UAS-GULP1 lines in Drosophila

The full length human GULP1 cDNA was amplified by PCR and subcloned into GAL4-responsive pUAST expression vector, and microinjected into Drosophila embryos (BestGene Inc, USA). The expressions of GULP1 in 5 independent lines (1M, 2M, 3M 4M and 5M) were determined by Western blotting.

Drosophila stocks

Fly stocks and crosses were raised on standard cornmeal medium with 1.25% agar; 10.5% dextrose; 10.5% cornmeal and 2.1% yeast. Flies were kept in incubators (LMS Ltd., UK) maintained at 18 oC, 19oC, 25oC or 28oC as specified. elav-GAL4 (458), gmr-GAL4 (1104), UAS-APP695, UAS-BACE (33797) were obtained from Bloomington Drosophila Stock Center, USA. Virgin females for crosses were collected within 8 hours at 25oC or 16 hours at 18oC.

Western blot analysis

Fifteen fly heads were collected and homogenized in 75 µl 2X SDS sample buffer containing 100mM Tris, 4% SDS, 0.2% Bromophenol blue, 20% Glycerol and 15 μl/ml β-mercaptoethanol. Lysates were then boiled for 10 minutes and separated by SDS/PAGE gels. Protein on the gels was transferred to nitrocellulose blotting membrane (PALL) using a wet blotting system (Bio-Rad). Blots were probed with the following antibodies: Anti-GULP1 [7]; Anti-APP [48]; Anti-BACE [49]; Anti-actin A2103 (Sigma).


Human Aβ1-40 and Aβ1-42 in the fly heads were determined by using the human β40 and β42 ELISA kits (Millipore). In brief, 15 fly heads were homogenized in 50 µl ice-cold 1X RIPA buffer containing 50 mM Tris, 150 mM NaCl, 1% SDS, 1% NP-40, 0.5% sodium deoxycholate, pH 8.0 and Complete™ protease inhibitor (Roche). The homogenates were diluted 10-fold with 450 µl sample diluent and then cleared by centrifugation at 15,000 rpm for 5 minutes at 4oC. 200 µl of supernatant of each sample was added to ELISA plate with primary antibody. After overnight incubation at 4oC, the ELISA plate was washed with wash buffer. Streptavidin-peroxidase-conjugate was then added for colorimetric signal development at room temperature. The colorimetric reaction was stopped by adding stop solution, and signals were measured at 450 nm by using a microplate reader (Bio-Rad).

Pseudopupil assay

Pseudopupil assay was performed essentially as described previously [58, 59]. In brief, 5 days post-eclosion (dpe) fly eyes were examined under a light microscope (Olympus CX31) with a 60X oil objective. At least 200 ommatidia from at least 15 adult flies obtained from three independent crosses were used to calculate the average number of rhabdomeres per ommatidium.

Locomotor activity assay

Locomotor activity of the flies were determined at 20 dpe. In brief, group of 10 flies were placed at the bottom of a 15mL falcon tube. The number of flies that successfully climbed up a vertical distance of 8 cm or more was recorded. At least 80 flies of each genotype were analyzed in an experiment. Three independent experiment were performed.

Longevity assay

Groups of 15 flies were placed in a food vial. Dead flies were counted every 3 days. At least 70 flies were assayed for each genotype from three independent crosses.

ARF6 activation assay

ARF6 activation assay was performed as described previously [60] by an active ARF6 pull-down kit (Cell Biolabs). ARF6 was detected by a mouse anti-ARF6 supplied from the kit.

Statistical analysis

The Mann-Whitney rank sum test was performed to compare mean differences between the average numbers of rhabdomeres per ommatidium in pseudopupil assay. One-way ANOVA test with Tukey post-hoc analysis was performed for ELISA analysis and climbing assay. Log-rank (Mantel-Cox) Test was performed for survival assay. A P-value of less than 0.05 was considered statistically significant. Significance is indicated as *p < 0.05, **p < 0.01.

Author contributions

WYVC and ACK designed and performed the experiments. ACK, JCKN, HYEC and KFL conceived the study, designed the experiments and wrote the manuscript.


There is no conflict of interest.


This work was supported by funds from the Health and Medical Research Fund (Grant number: 03140086), Research Grants Council Hong Kong (Grant number: 469610) and CUHK direct grant scheme (Grant numbers: 4053045 and 4053102).


1. Liu QA, Hengartner MO. Human CED-6 encodes a functional homologue of the Caenorhabditis elegans engulfment protein CED-6. Curr Biol. 1999; 9: 1347-50.

2. Smits E, Van Criekinge W, Plaetinck G, Bogaert T. The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells. Curr Biol. 1999; 9: 1351-4.

3. Su HP, Nakada-Tsukui K, Tosello-Trampont AC, Li Y, Bu G, Henson PM, Ravichandran KS. Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP). J Biol Chem. 2002; 277: 11772-9.

4. Park SY, Kang KB, Thapa N, Kim SY, Lee SJ, Kim IS. Requirement of adaptor protein GULP during stabilin-2-mediated cell corpse engulfment. J Biol Chem. 2008; 283: 10593-600. doi: 10.1074/jbc.M709105200.

5. Kim S, Park SY, Kim SY, Bae DJ, Pyo JH, Hong M, Kim IS. Cross-talk between engulfment receptors, stabilin-2 and integrin alphavbeta5 orchestrates engulfment of phosphatidylserine exposed erythrocytes. Mol Cell Biol. 2012; 14: 2698-708. doi: 10.1128/MCB.06743-11.10.1128.

6. Park SY, Kim SY, Kang KB, Kim IS. Adaptor protein GULP is involved in stabilin-1-mediated phagocytosis. Biochem Biophys Res Commun. 2010; 398: 467-72. doi: 10.1016/j.bbrc.2010.06.101.

7. Hao CY, Perkinton MS, Chan WW, Chan HY, Miller CC, Lau KF. GULP1 is a novel APP-interacting protein that alters APP processing. Biochem J. 2011; 436: 631-9. doi: 10.1042/BJ20110145.

8. Beyer AS, von Einem B, Schwanzar D, Keller IE, Hellrung A, Thal DR, Ingelsson M, Makarova A, Deng M, Chhabra ES, Propper C, Bockers TM, Hyman BT, et al. Engulfment adapter PTB domain containing 1 interacts with and affects processing of the amyloid-beta precursor protein. Neurobiol Aging. 2012; 33: 732-43. doi: 10.1016/j.neurobiolaging.2010.06.006.

9. Taru H, Suzuki T. Regulation of the Physiological Function and Metabolism of AβPP by AβPP Binding Proteins. J Alzheimers Dis. 2009; 18: 253-65. doi: 10.3233/JAD-2009-1148.

10. Buoso E, Lanni C, Schettini G, Govoni S, Racchi M. beta-Amyloid precursor protein metabolism: focus on the functions and degradation of its intracellular domain. Pharmacol Res. 2010; 62: 308-17. doi: 10.1016/j.phrs.2010.05.002.

11. Shulman JM, Shulman LM, Weiner WJ, Feany MB. From fruit fly to bedside: translating lessons from Drosophila models of neurodegenerative disease. Curr Opin Neurol. 2003; 16: 443-9.

12. Moloney A, Sattelle DB, Lomas DA, Crowther DC. Alzheimer’s disease: insights from Drosophila melanogaster models. Trends Biochem Sci. 2010; 35: 228-35. doi: 10.1016/j.tibs.2009.11.004.

13. Iijima K, Liu HP, Chiang AS, Hearn SA, Konsolaki M, Zhong Y. Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer’s disease. Proc Natl Acad Sci U S A. 2004; 101: 6623-8. doi: 10.1073/pnas.0400895101.

14. Chakraborty R, Vepuri V, Mhatre SD, Paddock BE, Miller S, Michelson SJ, Delvadia R, Desai A, Vinokur M, Melicharek DJ, Utreja S, Khandelwal P, Ansaloni S, et al. Characterization of a Drosophila Alzheimer’s disease model: pharmacological rescue of cognitive defects. PLoS One. 2011; 6: e20799. doi: 10.1371/journal.pone.0020799.

15. Le Corre S, Klafki HW, Plesnila N, Hubinger G, Obermeier A, Sahagun H, Monse B, Seneci P, Lewis J, Eriksen J, Zehr C, Yue M, McGowan E, et al. An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice. Proc Natl Acad Sci U S A. 2006; 103: 9673-8. doi: 10.1073/pnas.0602913103.

16. Crowther DC, Kinghorn KJ, Miranda E, Page R, Curry JA, Duthie FA, Gubb DC, Lomas DA. Intraneuronal Abeta, non-amyloid aggregates and neurodegeneration in a Drosophila model of Alzheimer’s disease. Neuroscience. 2005; 132: 123-35. doi: 10.1016/j.neuroscience.2004.12.025.

17. Chan HY, Bonini NM. Drosophila models of human neurodegenerative disease. Cell Death Differ. 2000; 7: 1075-80.

18. Sang TK, Jackson GR. Drosophila models of neurodegenerative disease. NeuroRx. 2005; 2: 438-46. doi: 10.1602/neurorx.2.3.438.

19. Greeve I, Kretzschmar D, Tschape JA, Beyn A, Brellinger C, Schweizer M, Nitsch RM, Reifegerste R. Age-dependent neurodegeneration and Alzheimer-amyloid plaque formation in transgenic Drosophila. J Neurosci. 2004; 24: 3899-906. doi: 10.1523/JNEUROSCI.0283-04.2004.

20. Finelli A, Kelkar A, Song HJ, Yang H, Konsolaki M. A model for studying Alzheimer’s Abeta42-induced toxicity in Drosophila melanogaster. Mol Cell Neurosci. 2004; 26: 365-75. doi: 10.1016/j.mcn.2004.03.001.

21. Iijima K, Ando K, Takeda S, Satoh Y, Seki T, Itohara S, Greengard P, Kirino Y, Nairn AC, Suzuki T. Neuron-specific phosphorylation of Alzheimer’s beta-amyloid precursor protein by cyclin-dependent kinase 5. J Neurochem. 2000; 75: 1085-91.

22. Sannerud R, Declerck I, Peric A, Raemaekers T, Menendez G, Zhou L, Veerle B, Coen K, Munck S, De Strooper B, Schiavo G, Annaert W. ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1. Proc Natl Acad Sci U S A. 2011; 108: E559-68. doi: 10.1073/pnas.1100745108.

23. Ma Z, Nie Z, Luo R, Casanova JE, Ravichandran KS. Regulation of Arf6 and ACAP1 signaling by the PTB-domain-containing adaptor protein GULP. Curr Biol. 2007; 17: 722-7.

24. Sabe H, Hashimoto S, Morishige M, Ogawa E, Hashimoto A, Nam JM, Miura K, Yano H, Onodera Y. The EGFR-GEP100-Arf6-AMAP1 signaling pathway specific to breast cancer invasion and metastasis. Traffic. 2009; 10: 982-93. doi: 10.1111/j.1600-0854.2009.00917.x.

25. Thinakaran G, Koo EH. Amyloid precursor protein trafficking, processing, and function. J Biol Chem. 2008; 284: 29615-9. doi: 10.1074/jbc.R800019200.

26. Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, Ito K. Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron. 2006; 50: 855-67. doi: 10.1016/j.neuron.2006.04.027.

27. Jha A, Watkins SC, Traub LM. The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers. Mol Biol Cell. 2012; 23: 1742-64. doi: 10.1091/mbc.E11-11-0939.

28. Cuttell L, Vaughan A, Silva E, Escaron CJ, Lavine M, Van Goethem E, Eid JP, Quirin M, Franc NC. Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis. Cell. 2008; 135: 524-34. doi: 10.1016/j.cell.2008.08.033.

29. Cirrito JR, Kang JE, Lee J, Stewart FR, Verges DK, Silverio LM, Bu G, Mennerick S, Holtzman DM. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron. 2008; 58: 42-51. doi: 10.1016/j.neuron.2008.02.003.

30. Fuentes-Medel Y, Logan MA, Ashley J, Ataman B, Budnik V, Freeman MR. Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris. PLoS Biol. 2009; 7: e1000184. doi: 10.1371/journal.pbio.1000184.

31. MacDonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR. The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron. 2006; 50: 869-81. doi: 10.1016/j.neuron.2006.04.028.

32. Berger C, Renner S, Luer K, Technau GM. The commonly used marker ELAV is transiently expressed in neuroblasts and glial cells in the Drosophila embryonic CNS. Dev Dyn. 2007; 236: 3562-8. doi: 10.1002/dvdy.21372.

33. Wyss-Coray T, Loike JD, Brionne TC, Lu E, Anankov R, Yan F, Silverstein SC, Husemann J. Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat Med. 2003; 9: 453-7. doi: 10.1038/nm838.

34. Liu CC, Hu J, Zhao N, Wang J, Wang N, Cirrito JR, Kanekiyo T, Holtzman DM, Bu G. Astrocytic LRP1 Mediates Brain Abeta Clearance and Impacts Amyloid Deposition. J Neurosci. 2017; 37: 4023-31. doi: 10.1523/JNEUROSCI.3442-16.2017.

35. Eugenin J, Vecchiola A, Murgas P, Arroyo P, Cornejo F, von Bernhardi R. Expression Pattern of Scavenger Receptors and Amyloid-beta Phagocytosis of Astrocytes and Microglia in Culture are Modified by Acidosis: Implications for Alzheimer’s Disease. J Alzheimers Dis. 2016; 53: 857-73. doi: 10.3233/JAD-160083.

36. Marsh SE, Abud EM, Lakatos A, Karimzadeh A, Yeung ST, Davtyan H, Fote GM, Lau L, Weinger JG, Lane TE, Inlay MA, Poon WW, Blurton-Jones M. The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function. Proc Natl Acad Sci U S A. 2016; 113: E1316-25. doi: 10.1073/pnas.1525466113.

37. Yamamoto N, Fujii Y, Kasahara R, Tanida M, Ohora K, Ono Y, Suzuki K, Sobue K. Simvastatin and atorvastatin facilitates amyloid beta-protein degradation in extracellular spaces by increasing neprilysin secretion from astrocytes through activation of MAPK/Erk1/2 pathways. Glia. 2016; 64: 952-62. doi: 10.1002/glia.22974.

38. Xiao Q, Yan P, Ma X, Liu H, Perez R, Zhu A, Gonzales E, Burchett JM, Schuler DR, Cirrito JR, Diwan A, Lee JM. Enhancing astrocytic lysosome biogenesis facilitates Abeta clearance and attenuates amyloid plaque pathogenesis. J Neurosci. 2014; 34: 9607-20. doi: 10.1523/JNEUROSCI.3788-13.2014.

39. Ma CI, Martin C, Ma Z, Hafiane A, Dai M, Lebrun JJ, Kiss RS. Engulfment protein GULP is regulator of transforming growth factor-beta response in ovarian cells. J Biol Chem. 2012; 287: 20636-51. doi: 10.1074/jbc.M111.314997.

40. Caraci F, Battaglia G, Bruno V, Bosco P, Carbonaro V, Giuffrida ML, Drago F, Sortino MA, Nicoletti F, Copani A. TGF-beta1 pathway as a new target for neuroprotection in Alzheimer’s disease. CNS Neurosci Ther. 2011; 17: 237-49. doi: 10.1111/j.1755-5949.2009.00115.x.

41. Wang B, Hu Q, Hearn MG, Shimizu K, Ware CB, Liggitt DH, Jin LW, Cool BH, Storm DR, Martin GM. Isoform-specific knockout of FE65 leads to impaired learning and memory. J Neurosci Res. 2004; 75: 12-24.

42. Lee JH, Lau KF, Perkinton MS, Standen CL, Rogelj B, Falinska A, McLoughlin DM, Miller CC. The neuronal adaptor protein X11β reduces amyloid β-protein levels and amyloid plaque formation in the brains of transgenic mice. J Biol Chem. 2004; 279: 49099-104.

43. Lee JH, Lau KF, Perkinton MS, Standen CL, Shemilt SJ, Mercken L, Cooper JD, McLoughlin DM, Miller CC. The neuronal adaptor protein X11α reduces Aβ levels in the brains of Alzheimer’s APPswe Tg2576 transgenic mice. J Biol Chem. 2003; 278: 47025-9.

44. Ho A, Liu X, Sudhof TC. Deletion of Mint proteins decreases amyloid production in transgenic mouse models of Alzheimer’s disease. J Neurosci. 2008; 28: 14392-400. doi: 10.1523/JNEUROSCI.2481-08.2008

45. Saluja I, Paulson H, Gupta A, Turner RS. X11alpha haploinsufficiency enhances Abeta amyloid deposition in Alzheimer’s disease transgenic mice. Neurobiol Dis. 2009; 36: 162-8. doi: 10.1016/j.nbd.2009.07.006.

46. Saito Y, Sano Y, Vassar R, Gandy S, Nakaya T, Yamamoto T, Suzuki T. X11 proteins regulate the translocation of amyloid beta-protein precursor (APP) into detergent-resistant membrane and suppress the amyloidogenic cleavage of APP by beta-site-cleaving enzyme in brain. J Biol Chem. 2008; 283: 35763-71. doi: 10.1074/jbc.M801353200.

47. Sano Y, Syuzo-Takabatake A, Nakaya T, Saito Y, Tomita S, Itohara S, Suzuki T. Enhanced amyloidogenic metabolism of APP in the X11L-deficient mouse brain. J Biol Chem. 2006; 281: 37853-60.

48. Lau KF, McLoughlin DM, Standen C, Miller CC. X11α and X11β interact with presenilin-1 via their PDZ domains. Mol Cell Neurosci. 2000; 16: 557-65.

49. Angeletti B, Waldron KJ, Freeman KB, Bawagan H, Hussain I, Miller CC, Lau KF, Tennant ME, Dennison C, Robinson NJ, Dingwall C. BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper. J Biol Chem. 2005; 280: 17930-7.

50. Dunning CJ, Black HL, Andrews KL, Davenport EC, Conboy M, Chawla S, Dowle AA, Ashford D, Thomas JR, Evans GJ. Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11alpha by Src kinase regulates the trafficking of amyloid precursor protein. J Neurochem. 2016; 137: 518-27. doi: 10.1111/jnc.13571.

51. Chow WN, Ngo JC, Li W, Chen YW, Tam KM, Chan HE, Miller CC, Lau KF. Phosphorylation of FE65 Serine-610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer’s disease amyloid precursor protein processing. Biochem J. 2015; 470: 303-17. doi: 10.1042/BJ20141485.

52. Cole SL, Vassar R. The role of amyloid precursor protein processing by BACE1, the β-secretase, in Alzheimer disease pathophysiology. J Biol Chem. 2008; 283: 29621-5. doi: 10.1074/jbc.R800015200.

53. Nekhoroshkova E, Albert S, Becker M, Rapp UR. A-RAF kinase functions in ARF6 regulated endocytic membrane traffic. PLoS One. 2009; 4: e4647. doi: 10.1371/journal.pone.0004647.

54. Chen RQ, Yang QK, Lu BW, Yi W, Cantin G, Chen YL, Fearns C, Yates JR 3rd, Lee JD. CDC25B mediates rapamycin-induced oncogenic responses in cancer cells. Cancer Res. 2009; 69: 2663-8. doi: 10.1158/0008-5472.CAN-08-3222.

55. Cantin GT, Yi W, Lu B, Park SK, Xu T, Lee JD, Yates JR 3rd. Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis. J Proteome Res. 2008; 7: 1346-51. doi: 10.1021/pr0705441.

56. DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A. 2001; 98: 8850-5. doi: 10.1073/pnas.151261398.

57. Abbott A, Dolgin E. Failed Alzheimer’s trial does not kill leading theory of disease. Nature. 2016; 540: 15-6. doi: 10.1038/nature.2016.21045.

58. Tsoi H, Lau CK, Lau KF, Chan HY. Perturbation of U2AF65/NXF1-mediated RNA nuclear export enhances RNA toxicity in polyQ diseases. Hum Mol Genet. 2011; 20: 3787-97. doi: 10.1093/hmg/ddr297.

59. Chan WM, Tsoi H, Wu CC, Wong CH, Cheng TC, Li HY, Lau KF, Shaw PC, Perrimon N, Chan HY. Expanded polyglutamine domain possesses nuclear export activity which modulates subcellular localization and toxicity of polyQ disease protein via exportin-1. Hum Mol Genet. 2011; 20: 1738-50. doi: 10.1093/hmg/ddr049.

60. Cheung HN, Dunbar C, Morotz GM, Cheng WH, Chan HY, Miller CC, Lau KF. FE65 interacts with ADP-ribosylation factor 6 to promote neurite outgrowth. FASEB J. 2014; 28: 337-49. doi: 10.1096/fj.13-232694.

Creative Commons License All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.
PII: 20062