The X11alpha Protein Slows Cellular Amyloid Precursor Protein Processing and Reduces Abeta 40 and Abeta 42 Secretion*

Jean-Paul BorgDagger , Yunning Yang§, Mylène De Taddéo-BorgDagger , Ben MargolisDagger **, and R. Scott Turner§parallel Dagger Dagger

From the Dagger  Howard Hughes Medical Institute,  Department of Internal Medicine and Biological Chemistry, and § Department of Neurology, University of Michigan Medical Center, Ann Arbor, Michigan 48109 and parallel  Veterans Affairs Medical Center Geriatric Research, Education, and Clinical Center, Ann Arbor, Michigan 48105

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Abeta peptides, including Abeta 40 and Abeta 42-the major component of amyloid plaques in Alzheimer's disease brain. The phosphotyrosine binding (PTB) domain of X11 binds to a peptide containing a YENPTY motif found in the carboxyl terminus of APP. We have cloned the full-length X11 gene now referred to as X11alpha . Coexpression of X11alpha with APP results in comparatively greater levels of cellular APP and less APPs, Abeta 40, and Abeta 42 recovered in conditioned medium of transiently transfected HEK 293 cells. These effects are impaired by a single missense mutation of either APP (Y682G within the YENPTY motif) or X11alpha (F608V within the PTB domain), which diminishes their interaction, thus demonstrating specificity. The inhibitory effect of X11alpha on Abeta 40 and Abeta 42 secretion is amplified by coexpression with the Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulse-chase analysis demonstrates that X11alpha prolongs the half-life of APP from ~2 h to ~4 h. The effects of X11alpha on cellular APP and APPs recovery were confirmed in a 293 cell line stably transfected with APP. The specific binding of the PTB domain of X11alpha to the YENPTY motif-containing peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Abeta 40, and Abeta 42 in conditioned medium of transfected HEK 293 cells. X11alpha may be involved in APP trafficking and metabolism in neurons and thus may be implicated in amyloidogenesis in normal aging and Alzheimer's disease brain.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The finding of miliary amyloid plaques in brain parenchyma is classically recognized as a hallmark of Alzheimer's disease (AD)1 pathology, although the role of amyloid deposition in AD is controversial. Recent data of the effects of gene mutations linked to familial AD suggests that the deposition of amyloid plaque in brain may play a causal role in the cascades leading to dementia and the pathologic abnormalities seen in AD brain: the amyloid hypothesis of AD (1-3). The major components of amyloid plaque are Abeta peptides, including Abeta 40 and Abeta 42, derived by constitutive proteolytic cleavage of amyloid precursor protein (APP) encoded on human chromosome 21. APP is a type I cell surface protein with an extracellular region, a transmembrane region, and short intracellular carboxyl-terminal cytoplasmic region. The Abeta sequence encompasses half of the transmembrane domain and a short part of the extracellular domain of APP. Abeta 40 and Abeta 42 are released by beta - and gamma -secretase activities that cleave APP at the amino and carboxyl termini of Abeta , respectively. By this pathway, Abeta and soluble APP (APPsbeta ) are released into the extracellular space. Alternate cleavage of APP within the Abeta sequence by an alpha -secretase activity releases APPsalpha and precludes full-length Abeta formation. In nonneuronal cell lines such as HEK 293 and Chinese hamster ovary cells, secreted APP fragments are generated primarily via the alpha -secretase pathway, although some Abeta is generated and secreted by beta -/gamma -secretases, primarily in the endosomal/lysosomal pathway. In these cells, endocytosis of cell surface APP requires the Tyr-Glu-Asn-Pro-Thr-Tyr (YENPTY) motif found in its intracellular carboxyl terminus and is thus necessary for Abeta generation (4, 5).

The cytoplasmic region of APP containing the YENPTY motif interacts with the PTB/PI (phosphotyrosine binding-protein interaction) domain of X11alpha (6), Fe65 (7, 8), and their homologous genes X11-like and Fe65-like (9, 10). X11 and Fe65 are highly expressed in neurons and contain a PTB domain originally described in Shc (11, 12). The Shc PTB domain interacts with Psi XNPXpY motifs (where Psi  is hydrophobic, X is any amino acid, N is Asn, P is Pro, and pY is phosphotyrosine) found in tyrosine kinase receptors and other tyrosine-phosphorylated proteins. The PTB domain of Shc is likely involved in tyrosine kinase signal transduction cascades. However, the PTB domain is a more general protein-protein interaction domain found in several otherwise unrelated proteins such as X11, Fe65, Numb, and Disabled. Although the PTB domains of these proteins are homologous to Shc, they differ by binding to nonphosphorylated partners (13, 14). The function of the newly described PTB domains is now being examined. For example, the PTB domain of Numb is crucial for the differentiation of sensory organ precursors in Drosophila (15, 16). Although the PTB domain of X11alpha binds specifically to the YENPTY-containing region of APP, the functional significance of this interaction is unknown. Deletion of the last 18 amino acids of APP encompassing the YENPTY motif or mutation of the amino-terminal tyrosine of the YENPTY motif of APP to glycine (Y682G) impairs binding to APP. Likewise, mutation of X11 at position 608 (F608V), previously referred to as the F479V mutation in the nonfull-length protein, impairs X11alpha -APP interaction (6). The importance of these specific amino acid residues was confirmed by analysis of the crystal structure of the X11alpha PTB domain complexed to a peptide encompassing the YENPTY motif of APP (17).

Recently, we and others have identified a second X11 gene in humans. We refer to the newly isolated gene as X11beta (Fig. 1). The goal of this study was to functionally characterize the interaction between X11alpha or X11beta with APP. Coexpression of X11alpha with APP in human embryonic kidney (HEK) 293 cells results in comparatively greater levels of cellular APP (APPc), and less APPs, Abeta 40, and Abeta 42 recovered in conditioned medium of transiently transfected cells. These effects are 1) correlated with a prolonged half-life of APPc, 2) impaired by a single missense mutation of either APP (Y682G) or X11alpha (F608V), and 3) amplified by coexpression with the Swedish mutation of APP (APPswe; K595N/M596L) found in a pedigree of early onset familial AD. Thus, structural and functional data implicate a normal role for X11alpha in APP trafficking and metabolism via a specific protein-protein interaction.


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Fig. 1.   Schematic representation of X11 proteins. X11alpha and X11beta contain highly related PTB and PDZ domains (84 and 85% identity, respectively) and divergent amino termini. Point mutations, i.e. phenylalanine (F) mutated to valine (V), introduced within the PTB domains are indicated by arrows.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- Human embryonic kidney 293 were grown in Dulbecco's modified Eagle's medium containing 100 units of penicillin/ml and 100 µg of streptomycin sulfate/ml supplemented with 10% fetal calf serum.

Cell Transfection and Protein Extraction-- Cells were split one day before transfection (1 × 106 cells/6-cm plate) and transfected with 10 µg of DNA by the calcium phosphate procedure. After 48 h, cells were washed twice with phosphate-buffered saline and lysed in lysis buffer (50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA) supplemented with protease inhibitors (aprotinin, leupeptin, and phenylmethylsulfonyl fluoride). All constructs were cloned in pRK5 vector as described previously (6). The APP695 isoform was used exclusively in this study.

For [35S]methionine labeling, cells were incubated with methionine-deficient Dulbecco's modified Eagle's medium containing 100 µCi/ml for 15 min followed by a chase in complete Dulbecco's modified Eagle's medium. After washing the cells with phosphate-buffered saline, proteins were extracted with lysis buffer. Conditioned media of transfected cells were collected before lysis, and proteins were immunoprecipitated overnight with Karen or 6E10 antibodies at 4 °C. Bound proteins were recovered on protein A-agarose beads. After extensive washing with lysis buffer, proteins were separated by SDS-PAGE and detected by immunoblot or by PhosphorImager and autoradiography. Radiolabeled proteins detected by PhosphorImager were quantitated with ImageQuant software (Molecular Dynamics).

Antibodies and ELISA-- The anti-myc antibody 9E10 (Oncogene Science) at 1 µg/ml was used for immunoblotting. The 22C11 monoclonal antibody (Boeringer Mannheim) was directed against an epitope of the extracellular region of APP. The polyclonal antisera 369 was directed to the cytoplasmic carboxyl terminus of APP. Karen is a goat polyclonal antisera directed to the secreted amino-terminal domain of APP (18). The monoclonal antibody 6E10 (Senetek) was raised to Abeta 1-17. The Abeta sandwich ELISA was performed as described previously (19) using BAN50 as the capture antibody and either horseradish peroxidase-coupled BA-27 or BC-05 as the detection antibody for Abeta 40 or Abeta 42, respectively. BAN-50 is a monoclonal antibody specific for Abeta 1-10.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

APP Mutations Affected Recovery of APPs in Conditioned Medium-- The YENPTY motif in the intracellular carboxyl terminus of APP is involved in its cellular processing. For example, deletion of this motif results in greater recovery of APPsalpha in conditioned medium (4, 5). Either deletion of the YENPTY sequence or mutation of the amino-terminal tyrosine of the motif (APP Y682G) abrogates binding to the X11alpha PTB domain (6). Thus, we hypothesized that the APP Y682G mutation would recapitulate the effects of YENPTY deletion on APPsalpha recovery. HEK 293 cells were transiently transfected with APP, APP Y682G, or APPswe constructs. The APPswe double mutation resulted in comparatively greater Abeta and less APPsalpha recovery in conditioned medium. Comparable levels of APP expression were verified by immunoblot of cell lysates with the anti-APP antibody 369 (Fig. 2A, upper panel). APPsalpha in conditioned media was immunoprecipitated and detected by immunoblot with 6E10 (Fig. 2A, lower panel). The APP Y682G mutation resulted in greater release of APPsalpha in conditioned medium, similar to deletion of the YENPTY motif (Fig. 2A, lower panel). As expected, APPswe resulted in a decrease in APPsalpha in conditioned medium. Transfected HEK 293 cells were also labeled with [35S]methionine, and APPsalpha was recovered by immunoprecipitation with 6E10, separated by SDS-PAGE, and revealed by autoradiography (Fig. 2B). Similar to the results of Fig. 2A, APPsalpha release was markedly increased by the Y682G mutation, although comparable expression of APP was found in cell lysates (data not shown). Thus, the X11alpha PTB domain binding site in APP is functionally important for APPsalpha release. Interference with this interaction either by deletion of the carboxyl terminus or mutation of the YENPTY domain of APP resulted in greater APPsalpha release into conditioned medium.


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Fig. 2.   Mutation within the X11alpha PTB domain binding site of APP increases APPsalpha recovery in conditioned medium. A, HEK 293 cells were transiently transfected with pRK5 (vector) only, pRK5-APP, pRK5-APPswe (Swedish mutation of APP), or pRK5-APP Y682G. Conditioned media and cell lysates were collected 48 h after transfection. Proteins in cell lysates were separated by SDS-PAGE, and APP was detected by immunoblot with antibody 369 (upper panel). APPsalpha in conditioned media was immunoprecipitated with 6E10, separated by SDS-PAGE, and detected by immunoblot with 6E10 (lower panel). B, after transfection in the same conditions as Fig. 2A, proteins from HEK 293 cells were radiolabeled for 4 h with [35S]methionine. APPc and APPsalpha in conditioned media were immunoprecipitated with 6E10 and detected by SDS-PAGE and autoradiography. Comparable amounts of APP were found in cell lysates (not shown).

X11alpha Expression Impaired alpha -Secretase Processing of APP-- In HEK 293 cells APP is metabolized primarily by an alpha -secretase activity at the cell surface, resulting in APPsalpha secretion. Because a fraction of X11alpha protein is localized at the cell membrane, we hypothesized that X11alpha would influence APPsalpha release. HEK 293 cells were transiently cotransfected with APP and myc-tagged X11alpha constructs or control vector (Fig. 3A). APP and X11alpha in cell lysates were detected by immunoblot. APPsalpha in conditioned medium was immunoprecipitated with Karen and detected by immunoblot with 6E10. Coexpression of APP with X11alpha resulted in decreased recovery of APPsalpha in an X11alpha dose-dependent manner. X11alpha also resulted in a large increase of APP in cell lysates. Although APPsalpha in medium was barely detectable when transfected with 5 µg of the X11alpha construct, no further increase of APP in the cell lysate was detected compared with 1 µg of X11alpha . This might suggest that APP is being processed by a beta -secretase pathway. Conditioned media were immunoprecipitated with Karen, and bound proteins were detected by immunoblot with 22C11, an anti-APP antibody directed against all APPs species. The same decrease in APPs was documented with this antibody, ruling out this possibility (Fig. 3A). We speculate that the generation of APP may be decreased by high expression of X11alpha or that the APP level reaches a plateau in the cell. Cells were also cotransfected with APP mutations (Fig. 3B). Coexpression of 5 µg of X11alpha construct with either APP or APPswe resulted in far less APPsalpha in conditioned medium. This result was expected because APPswe does not influence X11alpha binding (data not shown). Coexpression of APP Y682G with X11alpha led only to a small decrease of APPsalpha compared with APP Y682G transfection only (Fig. 3B). Accordingly, APP Y682G retained only 5-10% of binding activity with X11alpha in vivo and in vitro (6). Collectively, this data suggested that the interaction of the PTB domain of X11alpha with the intracellular region of APP containing the YENPTY motif impaired release of APPsalpha .


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Fig. 3.   Coexpression of X11alpha with APP decreases recovery of APPsalpha in conditioned medium. A and B.,HEK 293 cells were transiently transfected with pRK5 (vector) only, pRK5-APP, pRK5-APPswe, or pRK5-APP Y682G in the absence or presence of pRK5 or pRK5-myc-X11alpha (1 or 5 µg of DNA). In panel B, 5 µg of pRK5-myc-X11alpha DNA was transfected. Proteins in cell extracts and conditioned medium were treated as in Fig. 2A. A c-myc epitope tag fused to the amino terminus of X11alpha allowed detection of this protein by anti-myc antibody. Proteins in cell lysates were detected by antibody 369 or anti-myc. Soluble APP in conditioned media was immunoprecipitated with Karen and detected by immunoblot with 6E10 for APPsalpha or 22C11 for total APPs.

X11alpha Coexpression with APP Decreased Abeta 40 and Abeta 42 Recovery in Conditioned Medium-- Abeta peptides, particularly Abeta 40 and Abeta 42, are also produced by constitutive APP metabolism. In contrast to alpha -secretase cleavage of APP, which precludes generation of full-length Abeta peptides, Abeta 40 and Abeta 42 are generated by beta - and gamma -secretase activities. In HEK 293 cells, Abeta peptides are generated almost exclusively by an endosomal pathway, which required a functional YENPTY motif (4). We assessed the effect of X11alpha coexpression with APP on Abeta 40 and Abeta 42 recovery in conditioned medium (Fig. 4), as measured by a sensitive and specific ELISA (19). Compared with cells transfected with X11alpha , transfection with APP resulted in measurable Abeta concentrations in medium. As expected, transfection with APPswe resulted in much greater levels of Abeta in conditioned medium (Fig. 4), in parallel with diminished APPs levels (Fig. 2). Conversely, the APP Y682G mutation resulted in a slight decrease in Abeta 40 and Abeta 42 release (Fig. 4A), in parallel with a slight increase in APPsalpha (Fig. 2). Coexpression of X11alpha with APP reduced the levels of Abeta 40 and Abeta 42 in medium. This inhibitory effect of X11alpha was amplified by coexpression with the APPswe mutation. As predicted by the binding data, APP Y682G metabolism was resistant to X11alpha effects.


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Fig. 4.   Coexpression of X11alpha with APP decreases recovery of Abeta peptides in conditioned medium. A and B, Abeta 40 and Abeta 42 were detected by ELISA of conditioned media of transiently transfected HEK 293 cells. Data represent absorbance minus background (vector only) converted to fmol/ml/h, based on a 40-h collection after transfection. The data plotted represent the mean + S.E. of 5-7 experiments for A and 4 experiments for B.

Thus, mutation of APP within the YENPTY motif impaired the inhibitory effects of X11alpha on Abeta secretion. We also determined the effect of coexpression of APP or APPswe with the mutation X11alpha F608V. This mutation lies within the carboxyl-terminal alpha -helix of X11alpha PTB domain and is a critical residue for PTB domain interaction (6). HEK 293 cells were cotransfected with APPswe and X11alpha constructs. As predicted from the binding data, the inhibitory effect of X11alpha on Abeta secretion was attenuated by X11alpha F608V coexpression (Fig. 4B). Thus the specific interaction of X11alpha with APP appears to inhibit release of Abeta 40 and Abeta 42 into conditioned medium. The ELISA data was not corrected for level of APPc, because apparent effects on Abeta secretion would be artifactually magnified.

X11alpha Coexpression Stabilized Cellular APP-- Our data suggests that X11alpha coexpression blocks the production of soluble APP metabolites and results in apparent greater levels of cellular APP, thus appearing to stabilize APP in the cell (Fig. 3). To test this hypothesis, we performed a pulse-chase analysis of HEK 293 cells transiently transfected with APP alone or with X11alpha . Cells were labeled for 15 min with [35 S]methionine before chase for up to 8 h. APP in cell lysates was immunoprecipitated by Karen antibody, separated by SDS-PAGE, and detected by PhosphorImager analysis and autoradiography. Radiolabeled APP was quantitated by ImageQuant software. Similar to previous reports, APP had a half-life of ~2 h in HEK 293 cells (Fig. 5). Coexpression of X11alpha prolonged APP half-life to ~4 h. To further evaluate the stabilization of cellular APP by X11alpha , we transiently expressed X11alpha in HEK 293 cells stably expressing APP and measured APP levels in cell lysates. In these experiments, X11alpha expression resulted in an apparent increase in the amount of APP in cell lysates. As predicted, the X11alpha F608V mutation had less effect (Fig. 6A, lower panel). Comparable amounts of X11alpha and X11alpha F608V were expressed in the cells (Fig. 6A, upper panel).


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Fig. 5.   X11alpha coexpression prolongs the half-life of cellular APP. A, HEK 293 cells were transiently transfected with pRK5-APP alone or in combination with X11alpha . After a pulse of 15 min with [35S]methionine, cells were washed with phosphate-buffered saline and chased with complete Dulbecco's modified Eagle's medium. Cellular extracts were prepared at 0, 1, 2, 4, and 8 h chase, and proteins were immunoprecipitated with Karen (anti-APP), separated by SDS-PAGE, and detected by PhosphorImager. B, radiolabeled proteins were quantitated by ImageQuant software. The data represent the mean + S.E. of three separate experiments.


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Fig. 6.   Stabilization of cellular APP by X11alpha or X11beta coexpression. A and B, HEK 293 cells stably transfected with APP were transiently transfected with pRK5 (vector) only, pRK5-myc-X11alpha , pRK5-X11alpha F608V, pRK5-myc-X11beta , or pRK5-myc-X11beta F519V. In Fig. 5B, 5 µg of DNA was transfected. Equal amounts of proteins from cell lysates were separated by SDS-PAGE and subject to immunoblot. The X11 proteins were detected by anti-myc antibody (upper panels), and cellular APP was revealed by antibody 369.

During the course of these studies with X11alpha , we cloned a second human X11 gene we named X11beta . This gene is located on chromosome 15 (human genome project) and encodes a 120-kDa protein with a predicted topology as X11alpha . Both X11alpha and X11beta are highly expressed in brain.2 The PTB and PDZ domains of X11beta were 80-90% identical to X11alpha domains and were thus predicted to bind similar targets. However, their amino termini were quite divergent. Accordingly, the X11beta PTB domain bound efficiently to APP in vitro and in cells in culture (data not shown) and had similar results on APP processing, i.e. diminished soluble metabolites and stabilization of APP in cells. Hence, expression of X11beta in HEK cells stably transfected with APP notably increased the amount of APPc (Fig. 6B). This effect is almost completely abolished by a mutation of X11beta analogous to X11alpha F608V. Taken together, these data demonstrate that both X11alpha and X11beta inhibit APP metabolism when overexpressed in HEK 293 cells.

    DISCUSSION
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Nonneuronal cell lines are instructive model systems of APP trafficking and metabolism. HEK 293 cells produce Abeta , primarily Abeta 40, via beta -/gamma -secretase activities in an endosomal pathway, although the primary metabolic products of APP in this cell line result from alpha -secretase activity (4). The intracellular carboxyl-terminal domain of APP, in particular the YENPTY consensus sequence required for endocytosis by clathrin-coated pits, plays an important role in APP processing and Abeta generation by the endosomal pathway (4, 5). X11alpha , a protein highly expressed in neurons, specifically interacts with a peptide encompassing the YENPTY motif of APP (6). We now demonstrate that the interaction of X11alpha or X11beta with APP has significant effects on its metabolism in HEK 293 cells and has implied effects on cellular trafficking of APP.

Coexpression of X11alpha with APP decreased the recovery of its soluble fragments APPsalpha , Abeta 40, and Abeta 42 in conditioned medium of HEK 293 cells. These effects are specific, as demonstrated by the use of a single point mutation within either the cytoplasmic YENPTY motif of APP or the PTB domain of X11alpha , which impairs their interaction and thus, X11alpha effects. The decreased recovery of soluble APP fragments in conditioned medium was observed in parallel with an apparent increase in APP in cell lysates. This suggested prolongation of the half-life of APPc, which was confirmed by pulse-chase analysis. Expression of a second member of the X11 gene family, i.e. X11beta , in HEK 293 cells had similar effects on APP processing. X11beta bound as efficiently as X11alpha to APP in vivo and in vitro (data not shown). Thus, the specific interaction of the X11 PTB domain with the YENPTY motif-containing region of APP appeared to retard its processing and prolong its half-life, resulting in decreased recovery of soluble proteolytic fragments. When coexpressed with APP, X11alpha slowed both the alpha -secretase pathway and the endosomal/lysosomal pathway leading to Abeta generation. The mechanisms and intracellular site(s) of X11alpha effects on APP metabolism are unknown, but one may hypothesize that X11alpha slows endosomal trafficking of APP. Alternatively, X11alpha may prevent the secretion of APPs, leading to an accumulation of APP in the cell. Interestingly, recent evidence suggests increased neuronal endocytosis and thus increased Abeta secretion in neurons of sporadic AD brain compared with age-matched control brain (20).

The observed effects of X11alpha on inhibition of Abeta secretion with APP coexpression are qualitatively similar but amplified by coexpression with APPswe. In effect, coexpression of X11alpha with APPswe decreased Abeta 40 and Abeta 42 secretion to that seen with APP expression only. Similar to APP, when coexpressed with APPswe, X11alpha retarded both the alpha -secretase pathway and Abeta generation. The Swedish mutation of APP promoted its metabolism by beta -/gamma -protease activities, resulting in a 5-10-fold increase in Abeta 40 and Abeta 42 secretion, with a concomitant decrease in the alpha -secretase pathway (21, 22). There are other important differences between APP and APPswe metabolism. For example, in contrast to APP, transfection of an APPswe construct lacking a cytoplasmic tail, which precludes reinternalization, did not reduce the secretion of Abeta peptides. Thus, an additional beta -/gamma -protease pathway in Golgi-derived vesicles or the Golgi itself is present in APPswe metabolism to Abeta in nonneuronal cells (23-26). X11alpha may affect metabolism of APPswe by this cellular pathway as well as the endosomal pathway of Abeta generation.

X11alpha and X11beta are neuronal proteins that contain two PDZ (PSD-95/Dlg/ZO-1) domains in addition to the PTB domain. The PDZ domains found in other neuronal membrane proteins such as the PSD-95 family and nitric oxide synthase are implicated in their membrane clustering and localization. Clustering and localization of proteins may serve to stabilize proteins and prolong their half-life (27). This is similar to the effects of X11 on APP that we observed in this study. Although the binding partners of the PDZ domain of X11alpha are unknown, a heterotrimeric complex of PDZ partner/X11alpha /APP may be implicated in X11alpha effects and APP localization. The Fe65 gene family is also expressed primarily in neurons, and the encoded protein contains a PTB domain that binds to APP (8) and thus may influence its metabolism. In addition to two PTB domains, Fe65 contains in its sequence a WW protein interaction domain that binds to proline-rich sequences (28). Thus, Fe65 and X11alpha may have differential effects on APP trafficking and metabolism based on the formation of alternate and potentially competing heterotrimeric complexes of APP with either a PDZ binding partner of X11alpha or a WW binding partner of Fe65.

Neuronal processing of APP is in some ways distinct from its metabolism in nonneuronal cells and results in greater Abeta generation compared with nonneuronal cells (29-32). In addition to having the more ubiquitous alpha -secretase pathway at or near the cell surface and endosomal/lysosomal processing of APP to Abeta , neuronal cells have additional beta /gamma processing of APP within the endoplasmic reticulum/early Golgi. This neuronal exocytic pathway favors the generation of a higher ratio of Abeta 42 to Abeta 40 compared with the beta -/gamma - proteases of the endocytic pathway (31, 18, 33). Interestingly, presenilins are localized primarily to the endoplasmic reticulum and Golgi (34-36), suggesting that presenilin-1 or presenilin-2 mutations linked to familial AD exert their effect on APP metabolism, specifically increased Abeta 42 secretion, by promotion of beta -/gamma -cleavage within this exocytic pathway. The effects of X11 on the metabolism of APP717 mutations or on APP metabolism coexpressed with presenilin mutations, all of which result in a higher ratio of Abeta 42 to Abeta 40 secretion (19, 37), are unknown.

It will be of interest to probe the effects of X11 coexpression in transgenic mice harboring mutations of human APP linked to familial AD. With aging, these transgenic animals develop a partial AD-like phenotype, in particular behavioral changes and amyloid deposition in brain (38-40). Examination of X11 and Fe65 expression and their binding partners in normal aging and AD brain may shed light on two unanswered questions in AD research, namely, the selective anatomic localization of amyloid plaques in brain and the increased risk of AD with aging. Finally, if the amyloid hypothesis of AD proves tenable, knowledge of the effects of X11 and Fe65 on APP metabolism may serve as the basis for novel therapeutic strategies to delay the onset or slow the progression of amyloid formation and thus the clinical dementia of AD.

    ACKNOWLEDGEMENTS

We thank Dr. N. Suzuki, Takeda Chemical Co., Japan, for antibodies BAN-50, BA-27, and BC-05 and Drs. B. Greenberg and S. Gandy for the antisera Karen and 369, respectively.

    FOOTNOTES

* The work was supported by a pilot of National Institutes of Health Grant P50 AG08671.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF047347 and AF047348.

Dagger Dagger To whom correspondence and reprint requests should be addressed: VAMC GRECC, 2215 Fuller Rd., Ann Arbor, MI 48105. Tel.: 734-761-7686; Fax: 734-761-7489; E-mail: raymondt{at}umich.edu.

** An investigator of the Howard Hughes Medical Institute.

1 The abbreviations used are: AD Alzheimer's disease; Abeta , amyloid-beta protein; APPc, cellular APP; APPswe, Swedish mutation of APP; APP, beta -amyloid precursor protein; APPsalpha , soluble APP cleaved by alpha -secretase, APPsbeta , soluble APP cleaved by beta -secretase; PDZ, PSD-95/Dlg/ZO-1; PI, protein interaction; PTB, phosphotyrosine binding; HEK, human embryonic kidney cells; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

2 J.-P. Borg and B. Margolis, unpublished data.

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Abstract
Introduction
Procedures
Results
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References

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