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* Department of Neurosciences, University of California, San Diego, La Jolla, California, USA; and the
Buck Institute for Age Research, Novato, California, USA
1Correspondence: Department of Neurosciences, University of California, San Diego, 9500 Gilman Dr., Mail Code 0691, La Jolla, CA 92093-0691, USA. E-mail: edkoo{at}ucsd.edu
SPECIFIC AIMS
We recently proposed a model of Aßbeta; toxicity wherein Aßbeta; accelerates the multimerization of APP in a manner similar to other known physiological pathways, such as Fas ligand (FasL)-induced cell death and cytokine receptor signaling. The aim of the study was to elucidate how Aßbeta; associates with APP to increase the susceptibility of neurons to cell death.
PRINCIPAL FINDINGS
1. The Aßbeta; effect on cell death is mediated through interaction with the cognate Aßbeta; region on APP
Amyloid ßbeta;-peptide (Aßbeta;) is postulated to play a central role in the pathogenesis of Alzheimer’s disease. Many varied mechanisms have been proposed for Aßbeta; induced toxicity but no consensus has emerged to account for its deleterious effects. We recently proposed that APP-dependent Aßbeta; toxicity involves the facilitation of APP complex formation by Aßbeta;. In this study we showed that Aßbeta; interacts directly and specifically with APP to facilitate APP homo-oligomerization by binding to its homologous sequences present at the cell surface.
First, when an ER retention signal (-KKQN-) was appended to APP, this APP-KK construct was largely excluded from the cell surface, but more importantly, Aßbeta; toxicity was largely attenuated as compared to expression of wild-type APP. This suggested that APP must be on the cell surface to facilitate Aßbeta; toxicity.
Second, C99, the APP C-terminal fragment formed following ßbeta;-secretase cleavage not only was able to form Aßbeta; induced complexes, but also potentiated Aßbeta; toxicity indistinguishable to full-length APP. This indicated that the critical domain in APP mediating Aßbeta; cytotoxicity was within the Aßbeta; sequence and the transmembrane domain, as neither the large extracellular region up to the ßbeta;-secretase site nor the cytoplasmic domain was necessary.
Third, we demonstrated that the 28 amino acid juxtamembrane residues (residues 597–624) present on the cell surface contained within C99 and APP695 are necessary and sufficient to enhance Aßbeta; toxicity. This was accomplished by expressing several APP chimeric molecules in which this region was exchanged with sequences from APLP2, where there was no primary sequence homology. Neither the chimeric C99 nor chimeric APP695 was able to augment Aßbeta; toxicity (Fig. 1
). Neither chimeric construct was able to form complexes by coimmunoprecipitation on Aßbeta; treatment.
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Fourth, to show that Aßbeta; interacted with its own cognate sequences in APP, we performed a ligand blotting experiment in which Aßbeta; was immobilized to nitrocellulose membrane and incubated with various radiolabeled APP constructs. This way we were also able to show that there is direct binding between Aßbeta; and APP. The binding is dependent on the aggregation state of Aßbeta;, as heavily aggregated Aßbeta; peptide failed to bind to APP.
2. The GYENPTY region is essential for Aßbeta;-induced cell death
We previously demonstrated that APP dimerization is associated with increased susceptibility to cell death. We have hypothesized that APP complex formation results in caspase activation to cleave APP at position Asp664 and the release of C31 peptide. To understand the mechanism whereby APP dimerization can initiate a cell death signal, we performed deletion and alanine mutagenesis through the -GYENPTY- motif in the APP cytoplasmic tail (residues 681–687). The rationale for focusing on this region is because this is a site that interacts with a number of cytoplasmic proteins, including Fe65, X11/Mint1, Jip-1b, and mDab1, and contained within C31.
When the GYENPTY domain was deleted from the APP cytoplasmic domain, we found that the APP-dependent component of Aßbeta; toxicity was essentially abrogated (Fig. 2
A). This was confirmed by alanine scanning mutagenesis through the GYENPTY motif, where each alanine mutant significantly attenuated cell death (Fig. 2C
).
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Second, we showed that this domain is necessary for cell death but is not necessary for either APP dimerization or cleavage at position 664 (Fig. 2B, D
). This indicated that the cytotoxic properties of C31 require a fully intact peptide, especially this seven amino acid motif. Finally, the importance of this region in cell death was confirmed by the lack of toxicity following expression of a C31 construct that lacks the GYENPTY domain.
CONCLUSIONS AND SIGNIFICANCE
In this study we showed that Aßbeta; interacts with the cognate Aßbeta; domain in APP present on the cell surface. In this way Aßbeta; may act as a ligand to its own precursor to enhance APP complex formation. It has been reported that Aßbeta; interacts with NH2 terminus of APP between residues 18–119. Consequently, it appears that Aßbeta; associates with APP through two distinct regions: in the NH2 terminus and with its own cognate domain at residues 597–624. Subsequent to APP multimerization, we have proposed that there is recruitment of cytosolic molecules to the complex, some involved in cleavage of APP, but others presumably transduce a cell death-related signal. Our studies show that the -GYENPTY- motif in the cytoplasmic domain is required for enhancing Aßbeta; toxicity. This region is not required for cleavage of APP, indicating that C31 may transmit its cytotoxicity via interaction with other molecules via this domain (Fig. 3
).
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Our studies also indicated that APP dimerization can occur through two apparently independent steps. APP complex formation can be induced by Aßbeta; in a pathway that requires the cognate Aßbeta; region within APP. In addition, both APP and C99 demonstrated a basal level of oligomerization in the absence of Aßbeta;, as seen from coimmunoprecipitation studies. This propensity for dimerization has been noted by others, although the dimerization site has not been mapped. However, the fact that both the APP-APLP2 and C99-APLP2 chimeras were able to form complexes even though the Aßbeta; sequence was largely absent indicated that basal APP dimerization was mediated by either the transmembrane or the cytoplasmic region.
We have suggested that a component of Aßbeta; toxicity is APP dependent, and this requires an intact caspase cleavage site in the cytoplasmic domain at position 664 of APP. Forced dimerization of APP led to increased susceptibility to cell death. In this study we demonstrated that the GYENPTY domain is critical for Aßbeta;-induced cell death such that loss of this region in APP abrogated Aßbeta; cytotoxicity. Loss of this region in C31 also attenuated C31 induced toxicity. It has been well established that the GYENPTY domain is a site that interacts with a number of cytosolic proteins. Moreover, interaction of APP with Fe65 through this domain is necessary for signal transduction mediated by the APP intracellular domain (AICD) formed after 
-secretase cleavage. It is unclear why this domain is critical for cytotoxicity. On the one hand, it has been reported that expression of an artificial AICD construct encompassing the C-terminal 58 residues of APP resulted in apoptosis in H4 glioma cells. Therefore, it is possible that C31, which is contained entirely within C58, induces cell death simply through a C58-mediated pathway. The latter was shown to require Tip60, possibly through a mechanism involving AICD-Tip60 complex formation and translocation into the nucleus. However, this pathway did not require either Fe65 or the GYENPTY domain, because mutagenesis of either tyrosine residues had no effect on cell death. Our results are more consistent with a proposed pathway in which AICD (C59) or C31 forms a ternary complex with Fe65 and CP2/LSF/LBP1 to induce expression of GSK-3ßbeta;, which in turn was correlated with apoptosis. However, both C58 and C59 are likely nonphysiologic or at least represent very minor species, because 
-cleavage of APP generates AICD 49 to 50 residues in length. Therefore, findings of cell death derived from the overexpression of C58 and C59 may be misleading.
The model we have presented here suggests that cleavage of APP at position 664 with release of C31 is associated with cell death. C31 has been difficult to detect, likely because of its short half-life or that it rapidly interacts with other cellular proteins. However, it is noteworthy that caspase-cleaved APP N-terminal fragment is quite stable and has been detected in brains of AD individuals and indeed, this fragment co localizes with senile plaques and neuronal cytoplasmic inclusions known as granulovacuolar degeneration. Thus, it has been proposed that this cleavage may represent an early event in AD pathogenesis. If the latter speculation is true, then it will be important to understand what regulates the caspase cleavage of APP and mediates the toxicity of the cleaved fragments, whether through a pathway that involves C31 or protein binding partners that interact with the GYENPTY domain of APP.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5032fje
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GYENPTY constructs were transiently expressed in N2a cells and the lysates immunoprecipitated with Flag antibody (Ab) followed by Western blot with anti-hemagglutinin Ab. APP complex formation in the APP-GYENPTY mutant was increased 
2-fold (1.8±0.26) (n=5) after Aßbeta; treatment, comparable to control APP (1.6±0.21) (n=5). Protein expression control is shown in the lower panel. C) Expression of single Alanine point mutants through the -GYENPTY- region in N2a showed reduction in Aßbeta; induced cell death (*P<0.001, 1-way ANOVA; post hoc Tukey) similar to the deletion mutant shown in panel A. Control for protein expression of the various APP constructs shown in the lower panel. D) APP is cleaved following the D664 residue after Aßbeta; treatment in the APP deletion mutant construct. Cleavage was assayed in B103 cells after transient transfection with APP, APP 
664 (upper panel), against the neoepitope at position 664 after cleavage. As shown, both APP and APP deletion mutant showed cleavage after Aßbeta; treatment. The C-terminal truncated APP cleavage fragment comigrates with the control APP