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FoxO mediates APP-induced AICD-dependent cell death

The amyloid precursor protein (APP) is a broadly expressed transmembrane protein that has a significant role in the pathogenesis of Alzheimer's disease (AD). APP can be cleaved at multiple sites to generate a series of fragments including the amyloid β (Aβ) peptides and APP intracellular domain (AICD). Although Aβ peptides have been proposed to be the main cause of AD pathogenesis, the role of AICD has been underappreciated. Here we report that APP induces AICD-dependent cell death in Drosophila neuronal and non-neuronal tissues. Our genetic screen identified the transcription factor forkhead box O (FoxO) as a crucial downstream mediator of APP-induced cell death and locomotion defect. In mammalian cells, AICD physically interacts with FoxO in the cytoplasm, translocates with FoxO into the nucleus upon oxidative stress, and promotes FoxO-induced transcription of pro-apoptotic gene Bim. These data demonstrate that APP modulates FoxO-mediated cell death through AICD, which acts as a transcriptional co-activator of FoxO.

cognitive decline leading to social and occupational debilitation, has been regarded as the most prevalent neurodegenerative disease in the world. 1 AD is pathologically differentiated from other causes of dementia by a reduction in synaptic contact, 2 accumulation of neurofibrillary tangles, 3,4 aggregates of the hyper-phosphorylated microtubule-associated protein tau, 5 and extracellular senile plaques (SPs) containing the amyloid b peptide (Ab). 6 Although senior individuals often develop some plaques and tangles as a consequence of aging, the brains of AD patients have a greater number of them in specific brain regions such as the temporal lobe. 3 The Ab peptide found predominantly in the SPs is generated from a large single-pass transmembrane protein termed as amyloid precursor protein (APP). 7 Intriguingly, mutations in APP have been implicated in familial susceptibility to AD. 8,9 APP can be cleaved at different sites to generate a series of fragments, including the Ab peptides, N-APP and APP intracellular domain (AICD). The extracellular SPs containing the Ab peptides have been postulated as a major cause of AD, 10,11 yet no direct correlation between Ab plaques load in the brain of AD patients and the severity of clinical symptoms has been documented. 12 On the other hand, Ab peptides were also proposed to aggregate into plaques to provide a protective mechanism by sequestering excess Ab oligomers released from high neuronal activity. 13 Ectopic expression of APP could induce neuronal cell death, but its mechanism has remained unclear. 14 A recent study suggested that, upon deprivation of trophic factors, APP is cleaved by b-secretase and an unknown secretase to release N-APP, which binds to the death receptor 6 and induces caspase-dependent neurodegeneration. 15 AICD was reported to form a transcription complex with Fe65 and histone acetyltransferase Tip60 to regulate gene expression, 16 and is required for kinesin-mediated axonal transport in vivo. 14 Despite many studies focusing on the pathological role of Ab in neurodegeneration, the function of AICD in development and AD pathogenesis has remained largely elusive.
The forkhead box O (FoxO) transcription factors, initially identified at the chromosomal breakpoints in rhabdomyosarcomas and acute leukemia, [17][18][19] contain the forkhead DNA-binding domain that belongs to the O subfamily. 20 The FoxO proteins have been highly conserved during evolution. Caenorhabditis elegans and Drosophila encode only one FoxO protein, named as Daf-16 21 and dFoxO, 22 respectively. Four FoxO proteins, FOXO1, FOXO2, FOXO3a, and FOXO4, have been identified in human, 23 and an additional member, FOXO6, was reported in mouse. 24 FoxO proteins are known to modulate various physiological processes including cell proliferation and differentiation, apoptosis, metabolism, oxidative stress, and lifespan. 22,[25][26][27] Although studies have confirmed a major role of FoxO in modulating cellular response to oxidative stress, a crucial factor involved in the pathogenesis of AD, a direct connection between FoxO proteins and APP, has not been established before.
Drosophila has been previously used as an animal model to study the pathogenesis of AD, but most studies have been focused on the neurotoxic effect of Ab peptides. In this work, we found that expression of human APP in Drosophila induces AICD-dependent cell death and locomotion defect, both of which were mediated by the transcription factor dFoxO. This mechanism has been conserved in mammalian cells, as AICDs could physically interact with FoxO proteins and act as a transcriptional co-factor to promote FoxOinduced expression of pro-apoptotic gene Bim.

Results and Discussion
APP induces cell death in Drosophila neuronal and non-neuronal tissues. To investigate the function of APP in animal development, we first expressed human APP (APP695) in the Drosophila nervous system by the panneuronal elav-Gal4 driver (Figure 1b To examine whether APP also induces cell death in tissues other than the nervous system, we expressed APP in the developing thorax driven by pnr-Gal4 (Figure 1n). Compared with the control (Figures 1o and o 0 ), expression of APP triggered cell death in the thorax region of third-instar larval wing disc (Figure 1p) and produced a small scutellum phenotype in the adult thorax (dashed line box, Figure 1p 0 ). A similar scutellum phenotype was also observed when APP expression was driven by ap-Gal4 (Supplementary Figure S1b). In addition, expression of APP (ptc4APP) along the anterior/posterior compartment boundary in the larval wing disc (Figure 2m) induced robust cell death ( Figure 2o) and produced a loss of the anterior cross vein phenotype in the adult wing (Figure 2b), as compared with the control (Figures 2a and n). Furthermore, expression of APP in the developing wing pouch driven by sd-Gal4 (sd4APP) provoked extensive cell death and generated a small wing phenotype in the adults (Supplementary Figure S2b). Together, these results demonstrate that APP is able to induce cell death in non-neuronal tissues during development. APP belongs to a conserved family that includes two additional amyloid precursor-like proteins, APLP1 and APLP2, which share with APP the intracellular domain (ICD), but lack the Ab domain. 28,29 These three proteins carry out essential overlapping functions in the development, as demonstrated by phenotypes of double or triple knock-out mice. [30][31][32][33] To examine whether Ab is required for APP-induced cell death, we expressed an APP/APLP2 chimeric protein containing the ICD domain, but not the Ab domain ( Figure 1a). This chimeric protein was able to recapitulate APP-induced cell death phenotypes  Figure S4). dfoxO D94 is a recently generated null allele that removes 420 kb of the dfoxO locus and produces no dFoxO protein, and hence, is stronger than all the previously reported dfoxo alleles. 38,39 Consistently, loss of dfoxO also suppressed ap4APPinduced scutellum phenotype (Supplementary Figures S1c and d) and sd4APP-induced small wing phenotype (Supplementary Figures S2c and d). On the other hand, ectopic expression of dFoxO or its human ortholog FoxO3a was able to phenocopy that of APP (Figures 2i-k and data not shown). Together, these data suggest that dFoxO is a crucial downstream factor that mediates APP-induced cell death in vivo.
To check whether dFoxO is required for APP-induced pathological defect, we examined the larval locomotion behavior. Compared with the control larvae that moved at a speed of about 430 mm/s (Figures 2s and t; Supplementary Video 1), expression of APP in the CNS (Appl4APP) resulted in enhanced cell death (data not shown) and considerably reduced moving ability (120 mm/s, Figures 2s and t; Supplementary Video 2). Expression of APP DCT failed to produce such locomotion defect (data not shown), suggesting AICD is essential for the pathological function of APP. We found that APP-induced locomotion defect was significantly suppressed by a mutation in dfoxO (330 mm/s,  Figures 2s and t), which by themselves had no obvious effect on locomotion (Figures  2s and t). Together, these results indicate that dFoxO is physiologically required for APP-induced behavioral defects.
To further verify the activation of dFoxO by APP in vivo, we examined the expression of pro-apoptotic gene hid, a wellknown transcriptional target of dFoxO. 40 We found that APP expression driven by sd-Gal4 in the wing pouch resulted in strong activation of hid transcription, as monitored by a hid-lacZ reporter, and reduced wing pouch size (Figures 2u and v). Both phenotypes were considerably suppressed in dfoxo heterozygous mutant discs (Figure 2w), indicating APPinduced hid activation is mediated by dFoxO. Consistently, expression of APP/APLP2 chimera (Supplementary Figure  S6c), but not that of APP DCT (Figure 2x), also induced hid transcription, confirming that the ICD domain, but not Ab, is essential for APP-induced FoxO activation.
AICD physically interacts with FoxO proteins in human cells. Our genetic work in Drosophila suggested that APP induces FoxO-mediated cell death through the release of AICD. To investigate the mechanism by which AICD promotes FoxO-mediate cell death, we co-expressed Myctagged AICD (AICD-Myc) with human FoxO1, FoxO3a, or FoxO4 in HEK 293T cells, and followed with co-immunoprecipitation assay. AICD was able to co-precipitate with FoxO1, FoxO3a, or FoxO4 (Figure 3a), and vice versa, FoxO3a or FoxO4 co-precipitated with AICD (Figure 3b), suggesting that AICD physically interacts with FoxO proteins in vivo. Consistently, AICD co-localized with FoxO predominantly in the cytoplasm of 293T cells (Figure 3e and Supplementary Figure S7). Treatment of cells with H 2 O 2 induced oxidative stress that promoted translocation of FoxO from the cytoplasm into the nucleus 41 (Figure 3f and Supplementary Figure S7). AICD also translocated into the nucleus under oxidative stress (Figures 3c and f,  Supplementary Figure S7), presumably through its interaction with FoxO proteins. In the absence of exogenous FoxO, AICD largely remained in the cytoplasm despite H 2 O 2 treatment (Figure 3c). Furthermore, AICD was able to translocate into the nucleus with FoxO4A3, a constitutive active form of FoxO4 that localized predominantly in the nucleus, 42 without oxidative stress challenge (Figures 3d  and i). Similar results for AICD and FoxO3a were also obtained in human neuro-blastoma SY-SH5Y cells (Figures  3g and h). On the other hand, the predominant localization of FoxO1 and FoxO3a in the cytoplasm and FoxO4A3 in the nucleus was not affected by the presence of AICD (Supplementary Figure S8).

AICD is a transcriptional cofactor of FoxO proteins.
Based upon the above results, we hypothesized that AICD could physically interact with FoxO proteins in the cytoplasm, FoxO mediates APP-induced cell death X Wang et al  To test this hypothesis, we examine the effect of AICD on the transcription of FoxO target genes. The pro-apoptotic gene Bim, which encodes a BH3-only protein that directly activates the cell death machinery, is a well-known transcriptional target of FoxO proteins. 43 In consistent with the hypothesis, AICD was able to upregulate Bim promoter-mediated transcription (Figure 4a), possibly through its interaction with the endogenous FoxO proteins. Furthermore, AICD cooperated with the constitutive active FoxO4A3 to synergistically activate luciferase expression driven by Bim promoter (Figure 4b) or 6 Â DBE (Figure 4c), a synthetic promoter containing six FoxO-responsive elements. 44 A similar cooperative effect with FoxO4A3 was observed for APPswe or C99 (Supplementary Figure S9) that comprises AICD, or the g-secretase PS1 (Supplementary Figure S10) that promotes AICD production from endogenous APP. Hydrogen peroxide treatment of cells stably expressing C99-GFP 45 resulted in enhanced AICD production accompanied with elevated Bim expression (lane 2, Figure 4d). On the other hand, AICD production was compromised by two g-secretase inhibitors, DBZ and Comp E, which resulted in significantly reduced Bim expression (lane 4 and 5, Figure 4d). Together, these data suggested that AICD acts as a transcriptional co-factor of FoxO to activate the expression of apoptotic gene Bim.

Conclusion
AD is one of the most prevalent neurodegenerative diseases in the world, although its underlying mechanism has remained largely elusive. In the present study, we analyzed the pathological functions of APP, an AD causative gene, in Drosophila, mouse and human cells. We demonstrated that ectopic expression of APP induces cell death and locomotion defect in Drosophila. Such function of APP depends on the production of AICD, and is mediated by the transcription factor dFoxO. Further studies in human cells confirmed that AICD interacts and co-localizes with FoxO proteins. Upon oxidative stress, AICD translocates from the cytoplasm into the nucleus together with FoxO, and functions as a transcriptional co-factor of FoxO to induce the transcription of Bim, a pro-apoptotic gene that activates the cell death machinery.
Our findings not only expand the existing body of knowledge about the physiological function of APP, but also shed light on discovering a potential cure for AD. Acridine orange staining. Brain, eye, and wing discs were dissected from the 3rd instar larvae in PBST and stained for acridine orange as described. 50 X-Gal staining. Wing discs were dissected from 3rd instar larvae in PBST and stained for b-galactosidase activity as described. 51 Larva locomotion test. Third instar larvae were collected and rinsed in 1% PBS, and subsequently transferred onto 3% agarose plate. The larvae were allowed to rest for 5 min before videotaping. The locomotion behaviors were analyzed by the Nikon software (NIS-Elements D, Nikon, Shanghai, China). Each genotype was tested with five larvae and each larva was tested for five times. For the track of the larvae, each genotype was recorded for 10 min and repeated for five times.

Materials and Methods
Plasmids. Expression vectors for GFP-FOXO1, GFP-FOXO3a, Flag-FOXO3a, and Bim-luciferase reporter were obtained from Z Yuan; 52 HA-FoxO4, HA-FoxO4A3, and 6 Â DBE-luciferase were obtained from B Burgering; 44   Immunofluorescence. Cells were transfected with the indicated plasmids for 24 h and then left untreated or treated with H 2 O 2 (200 mM, final concentration) for 1 h, fixed in 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Cells were incubated with the indicated primary antibodies followed by Alexa Fluor-conjugated secondary antibodies and observed under a fluorescent microscope (Olympus, Tokyo, Japan).
Immunoprecipitation and immunoblotting. Cells were transfected with the indicated plasmids for 24 h. Precleared lysates were incubated with the indicated antibodies followed by precipitation with protein G-Sepharose beads. Immune complexes were washed with lysis buffer, eluted in 2 Â SDS sample buffer, and subjected to western blot analysis using corresponding antibodies.
Luciferase reporter assay. HEK 293T cells were transfected with effector plasmids and luciferase reporters. pRL-TK Renilla luciferase construct was co-transfected as an internal control. Cells were harvested and subjected to dual-luciferase assay (Promega, Madison, WI, USA) after 24 h.

Cell Death and
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