The X11 Protein Slows Cellular Amyloid Precursor Protein Processing ...

THE JOURNAL

OF

BIOLOGICAL CHEMISTRY

Vol. 273, No. 24, Issue of June 12, pp. 14761–14766, 1998 Printed in U.S.A.

The X11a Protein Slows Cellular Amyloid Precursor Protein Processing and Reduces Ab40 and Ab42 Secretion* (Received for publication, February 10, 1998, and in revised form, April 7, 1998)

Jean-Paul Borg‡, Yunning Yang§, Myle`ne De Tadde´o-Borg‡, Ben Margolis‡¶**, and R. Scott Turner§i‡‡ From the ‡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 iVeterans Affairs Medical Center Geriatric Research, Education, and Clinical Center, Ann Arbor, Michigan 48105

Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Ab peptides, including Ab40 and Ab42–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 X11a. Coexpression of X11a with APP results in comparatively greater levels of cellular APP and less APPs, Ab40, and Ab42 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 X11a (F608V within the PTB domain), which diminishes their interaction, thus demonstrating specificity. The inhibitory effect of X11a on Ab40 and Ab42 secretion is amplified by coexpression with the Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulsechase analysis demonstrates that X11a prolongs the half-life of APP from ;2 h to ;4 h. The effects of X11a 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 X11a to the YENPTY motifcontaining peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Ab40, and Ab42 in conditioned medium of transfected HEK 293 cells. X11a 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.

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 * 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. This 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/EBI Data Bank with accession number(s) AF047347 and AF047348. ‡‡ 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: [email protected]. ** An investigator of the Howard Hughes Medical Institute. 1 The abbreviations used are: AD Alzheimer’s disease; Ab, amyloid-b protein; APPc, cellular APP; APPswe, Swedish mutation of APP; APP, b-amyloid precursor protein; APPsa, soluble APP cleaved by a-secretase, APPsb, soluble APP cleaved by b-secretase; PDZ, PSD-95/Dlg/ ZO-1; PI, protein interaction; PTB, phosphotyrosine binding; HEK, human embryonic kidney cells; PAGE, polyacrylamide gel electrophoreThis paper is available on line at http://www.jbc.org

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 Ab peptides, including Ab40 and Ab42, 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 Ab sequence encompasses half of the transmembrane domain and a short part of the extracellular domain of APP. Ab40 and Ab42 are released by b- and g-secretase activities that cleave APP at the amino and carboxyl termini of Ab, respectively. By this pathway, Ab and soluble APP (APPsb) are released into the extracellular space. Alternate cleavage of APP within the Ab sequence by an a-secretase activity releases APPsa and precludes full-length Ab formation. In nonneuronal cell lines such as HEK 293 and Chinese hamster ovary cells, secreted APP fragments are generated primarily via the a-secretase pathway, although some Ab is generated and secreted by b-/g-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 Ab generation (4, 5). The cytoplasmic region of APP containing the YENPTY motif interacts with the PTB/PI (phosphotyrosine binding-protein interaction) domain of X11a (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 CXNPXpY motifs (where C 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 X11a 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 encom-

sis; ELISA, enzyme-linked immunosorbent assay.

14761

14762

Analysis of X11-APP Interaction

FIG. 1. Schematic representation of X11 proteins. X11a and X11b 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.

passing 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 X11a-APP interaction (6). The importance of these specific amino acid residues was confirmed by analysis of the crystal structure of the X11a 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 X11b (Fig. 1). The goal of this study was to functionally characterize the interaction between X11a or X11b with APP. Coexpression of X11a with APP in human embryonic kidney (HEK) 293 cells results in comparatively greater levels of cellular APP (APPc), and less APPs, Ab40, and Ab42 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 X11a (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 X11a in APP trafficking and metabolism via a specific protein-protein interaction. EXPERIMENTAL PROCEDURES

Cell Culture—Human embryonic kidney 293 were grown in Dulbecco’s modified Eagle’s medium containing 100 units of penicillin/ml and 100 mg of streptomycin sulfate/ml supplemented with 10% fetal calf serum. Cell Transfection and Protein Extraction—Cells were split one day before transfection (1 3 106 cells/6-cm plate) and transfected with 10 mg 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 methioninedeficient Dulbecco’s modified Eagle’s medium containing 100 mCi/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 mg/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

FIG. 2. Mutation within the X11a PTB domain binding site of APP increases APPsa recovery in conditioned medium. A, HEK 293 cells were transiently transfected with pRK5 (vector) only, pRK5APP, 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). APPsa 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 APPsa 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). 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 Ab1–17. The Ab sandwich ELISA was performed as described previously (19) using BAN50 as the capture antibody and either horseradish peroxidasecoupled BA-27 or BC-05 as the detection antibody for Ab40 or Ab42, respectively. BAN-50 is a monoclonal antibody specific for Ab1–10. RESULTS

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 APPsa 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 X11a PTB domain (6). Thus, we hypothesized that the APP Y682G mutation would recapitulate the effects of YENPTY deletion on APPsa recovery. HEK 293 cells were transiently transfected with APP, APP Y682G, or APPswe constructs. The APPswe double mutation resulted in comparatively greater Ab and less APPsa 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). APPsa in conditioned media was immunoprecipitated and detected by immunoblot with 6E10 (Fig. 2A, lower panel). The APP Y682G mutation resulted in greater release of APPsa in conditioned medium, similar to deletion of the YENPTY motif (Fig. 2A, lower panel). As expected, APPswe resulted in a decrease in


FIG. 3. Coexpression of X11a with APP decreases recovery of APPsa 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-mycX11a (1 or 5 mg of DNA). In panel B, 5 mg of pRK5-myc-X11a 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 X11a 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 APPsa or 22C11 for total APPs.

APPsa in conditioned medium. Transfected HEK 293 cells were also labeled with [35S]methionine, and APPsa was recovered by immunoprecipitation with 6E10, separated by SDS-PAGE, and revealed by autoradiography (Fig. 2B). Similar to the results of Fig. 2A, APPsa release was markedly increased by the Y682G mutation, although comparable expression of APP was found in cell lysates (data not shown). Thus, the X11a PTB domain binding site in APP is functionally important for APPsa release. Interference with this interaction either by deletion of the carboxyl terminus or mutation of the YENPTY domain of APP resulted in greater APPsa release into conditioned medium. X11a Expression Impaired a-Secretase Processing of APP—In HEK 293 cells APP is metabolized primarily by an a-secretase activity at the cell surface, resulting in APPsa secretion. Because a fraction of X11a protein is localized at the cell membrane, we hypothesized that X11a would influence APPsa release. HEK 293 cells were transiently cotransfected with APP and myc-tagged X11a constructs or control vector (Fig. 3A). APP and X11a in cell lysates were detected by immunoblot. APPsa in conditioned medium was immunoprecipitated with Karen and detected by immunoblot with 6E10.

14763

Coexpression of APP with X11a resulted in decreased recovery of APPsa in an X11a dose-dependent manner. X11a also resulted in a large increase of APP in cell lysates. Although APPsa in medium was barely detectable when transfected with 5 mg of the X11a construct, no further increase of APP in the cell lysate was detected compared with 1 mg of X11a. This might suggest that APP is being processed by a b-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 X11a or that the APP level reaches a plateau in the cell. Cells were also cotransfected with APP mutations (Fig. 3B). Coexpression of 5 mg of X11a construct with either APP or APPswe resulted in far less APPsa in conditioned medium. This result was expected because APPswe does not influence X11a binding (data not shown). Coexpression of APP Y682G with X11a led only to a small decrease of APPsa compared with APP Y682G transfection only (Fig. 3B). Accordingly, APP Y682G retained only 5–10% of binding activity with X11a in vivo and in vitro (6). Collectively, this data suggested that the interaction of the PTB domain of X11a with the intracellular region of APP containing the YENPTY motif impaired release of APPsa. X11a Coexpression with APP Decreased Ab40 and Ab42 Recovery in Conditioned Medium—Ab peptides, particularly Ab40 and Ab42, are also produced by constitutive APP metabolism. In contrast to a-secretase cleavage of APP, which precludes generation of full-length Ab peptides, Ab40 and Ab42 are generated by b- and g-secretase activities. In HEK 293 cells, Ab peptides are generated almost exclusively by an endosomal pathway, which required a functional YENPTY motif (4). We assessed the effect of X11a coexpression with APP on Ab40 and Ab42 recovery in conditioned medium (Fig. 4), as measured by a sensitive and specific ELISA (19). Compared with cells transfected with X11a, transfection with APP resulted in measurable Ab concentrations in medium. As expected, transfection with APPswe resulted in much greater levels of Ab in conditioned medium (Fig. 4), in parallel with diminished APPs levels (Fig. 2). Conversely, the APP Y682G mutation resulted in a slight decrease in Ab40 and Ab42 release (Fig. 4A), in parallel with a slight increase in APPsa (Fig. 2). Coexpression of X11a with APP reduced the levels of Ab40 and Ab42 in medium. This inhibitory effect of X11a was amplified by coexpression with the APPswe mutation. As predicted by the binding data, APP Y682G metabolism was resistant to X11a effects. Thus, mutation of APP within the YENPTY motif impaired the inhibitory effects of X11a on Ab secretion. We also determined the effect of coexpression of APP or APPswe with the mutation X11a F608V. This mutation lies within the carboxylterminal a-helix of X11a PTB domain and is a critical residue for PTB domain interaction (6). HEK 293 cells were cotransfected with APPswe and X11a constructs. As predicted from the binding data, the inhibitory effect of X11a on Ab secretion was attenuated by X11a F608V coexpression (Fig. 4B). Thus the specific interaction of X11a with APP appears to inhibit release of Ab40 and Ab42 into conditioned medium. The ELISA data was not corrected for level of APPc, because apparent effects on Ab secretion would be artifactually magnified. X11a Coexpression Stabilized Cellular APP—Our data suggests that X11a 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

14764


FIG. 4. Coexpression of X11a with APP decreases recovery of Ab peptides in conditioned medium. A and B, Ab40 and Ab42 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 1 S.E. of 5–7 experiments for A and 4 experiments for B.

HEK 293 cells transiently transfected with APP alone or with X11a. 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 X11a prolonged APP half-life to ;4 h. To further evaluate the stabilization of cellular APP by X11a, we transiently expressed X11a in HEK 293 cells stably expressing APP and measured APP levels in cell lysates. In these experiments, X11a expression resulted in an apparent increase in the amount of APP in cell lysates. As predicted, the X11a F608V mutation had less effect (Fig. 6A, lower panel). Comparable amounts of X11a and X11a F608V were expressed in the cells (Fig. 6A, upper panel). During the course of these studies with X11a, we cloned a second human X11 gene we named X11b. This gene is located on chromosome 15 (human genome project) and encodes a 120-kDa protein with a predicted topology as X11a. Both X11a and X11b are highly expressed in brain.2 The PTB and PDZ domains of X11b were 80 –90% identical to X11a domains and were thus predicted to bind similar targets. However, their amino termini were quite divergent. Accordingly, the X11b PTB domain bound efficiently to APP in vitro and in cells in culture (data not shown) and had similar results on APP proc2

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

essing, i.e. diminished soluble metabolites and stabilization of APP in cells. Hence, expression of X11b 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 X11b analogous to X11a F608V. Taken together, these data demonstrate that both X11a and X11b inhibit APP metabolism when overexpressed in HEK 293 cells. DISCUSSION

Nonneuronal cell lines are instructive model systems of APP trafficking and metabolism. HEK 293 cells produce Ab, primarily Ab40, via b-/g-secretase activities in an endosomal pathway, although the primary metabolic products of APP in this cell line result from a-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 Ab generation by the endosomal pathway (4, 5). X11a, 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 X11a or X11b with APP has significant effects on its metabolism in HEK 293 cells and has implied effects on cellular trafficking of APP. Coexpression of X11a with APP decreased the recovery of its soluble fragments APPsa, Ab40, and Ab42 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 X11a,


14765

FIG. 6. Stabilization of cellular APP by X11a or X11b coexpression. A and B, HEK 293 cells stably transfected with APP were transiently transfected with pRK5 (vector) only, pRK5-myc-X11a, pRK5X11a F608V, pRK5-myc-X11b, or pRK5-myc-X11b F519V. In Fig. 5B, 5 mg 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. FIG. 5. X11a coexpression prolongs the half-life of cellular APP. A, HEK 293 cells were transiently transfected with pRK5-APP alone or in combination with X11a. 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 1 S.E. of three separate experiments.

which impairs their interaction and thus, X11a 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. X11b, in HEK 293 cells had similar effects on APP processing. X11b bound as efficiently as X11a 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, X11a slowed both the a-secretase pathway and the endosomal/lysosomal pathway leading to Ab generation. The mechanisms and intracellular site(s) of X11a effects on APP metabolism are unknown, but one may hypothesize that X11a slows endosomal trafficking of APP. Alternatively, X11a 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 Ab secretion in neurons of sporadic AD brain compared with agematched control brain (20).

The observed effects of X11a on inhibition of Ab secretion with APP coexpression are qualitatively similar but amplified by coexpression with APPswe. In effect, coexpression of X11a with APPswe decreased Ab40 and Ab42 secretion to that seen with APP expression only. Similar to APP, when coexpressed with APPswe, X11a retarded both the a-secretase pathway and Ab generation. The Swedish mutation of APP promoted its metabolism by b-/g-protease activities, resulting in a 5–10-fold increase in Ab40 and Ab42 secretion, with a concomitant decrease in the a-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 Ab peptides. Thus, an additional b-/g-protease pathway in Golgi-derived vesicles or the Golgi itself is present in APPswe metabolism to Ab in nonneuronal cells (23–26). X11a may affect metabolism of APPswe by this cellular pathway as well as the endosomal pathway of Ab generation. X11a and X11b 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 X11a are unknown, a heterotrimeric complex of PDZ partner/X11a/APP may be implicated in X11a effects and APP localization. The Fe65 gene family is also expressed primarily in neurons, and the encoded

14766


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 X11a 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 X11a 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 Ab generation compared with nonneuronal cells (29 –32). In addition to having the more ubiquitous a-secretase pathway at or near the cell surface and endosomal/lysosomal processing of APP to Ab, neuronal cells have additional b/g processing of APP within the endoplasmic reticulum/early Golgi. This neuronal exocytic pathway favors the generation of a higher ratio of Ab42 to Ab40 compared with the b-/g- 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 Ab42 secretion, by promotion of b-/gcleavage 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 Ab42 to Ab40 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. Acknowledgments—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. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9.

Hardy, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2095–2097 Selkoe, D. J. (1996) J. Biol. Chem. 271, 18295–18298 Selkoe, D. J. (1997) Science 275, 630 – 631 Haass, C., Hung, A. Y., Schlossmacher, M. G., Teplow, D. B., and Selkoe, D. J. (1993) J. Biol. Chem. 268, 3021–3024 Koo, E. H., and Squazzo, S. L. (1994) J. Biol. Chem. 269, 17386 –17389 Borg, J.-P., Ooi, J., Levy, E., and Margolis, B. (1996) Mol. Cell. Biol. 16, 6229 – 6241 Fiore, F., Zambrano, N., Minopoli, G., Donini, V., Duilio, A., and Russo, T. (1995) J. Biol. Chem. 270, 30853–30856 Zambrano, N., Buxbaum, J. D., Minopoli, G., Fiore, F., De Candia, P., De Renzis, S., Faraonio, R., Sabo, S., Cheetham, J., Sudol, M., and Russo, T. (1997) J. Biol. Chem. 272, 6399 – 6405 Guenette, S. Y., Chen, J., Jondro, P. D., and Tanzi, R. (1996) Proc. Natl. Acad.

Sci. U. S. A. 93, 10832–10837 10. McLoughlin, D. M., and Miller, C. C. J. (1996) FEBS Lett. 397, 197–200 11. Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B. (1994) J. Biol. Chem. 269, 32031–32034 12. Kavanaugh, W. M., and Williams, L. T. (1994) Science 266, 1862–1865 13. Borg, J.-P., and Margolis, B. (1998) Curr. Top. Microbiol Immunol. 228, 23–38 14. Chien, C.-T., Wang, S., Rothenberg, M., Jan, L. Y., and Jan, Y. N. (1998) Mol. Cell. Biol. 18, 598 – 607 15. Guo, M., Jan, L. Y., and Jan, Y. N. (1996) Neuron 17, 27– 41 16. Yaich, L., Ooi, J., Park, M., Borg, J.-P., Landry, C., Bodmer, R., and Margolis, B. (1998) J. Biol. Chem. 273, 10381–10388 17. Zhang, Z., Lee, C.-H., Mandiyan V., Borg, J.-P., Margolis, B., Schlessinger, J., and Kuriyan, J. (1997) EMBO J. 16, 6141– 6150 18. Chyung, A. S. C., Greenberg, B. D., Cook, D. G., Doms, R. W., and Lee, V. M.-Y. (1997) J. Cell. Biol. 138, 671– 680 19. Suzuki, N., Cheung, T. T., Cai, X.-D., Odaka, A., Otvos, L., Jr., Eckman, C., Golde, T. E., and Younkin, S. G. (1994) Science 264, 1336 –1340 20. Cataldo, A. M., Barnett, J. L., Pieroni, C., and Nixon, R. A. (1997) J. Neurosci. 17, 6142– 6151 21. Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672– 674 22. Cai, X.-D., Golde, T. E., and Younkin, S. G. (1993) Science 259, 514 –516 23. Haass, C., Lemere, C. A., Capell, A., Citron, P., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nat. Med. 1, 1291–1296 24. Martin, B. L., Schrader-Fischer, G., Busciglio, J., Duke, M., Paganetti, P, and Yankner, B. A. (1995) J. Biol. Chem. 270, 26727–26730 25. Esselmani, R., Macq, A.-F., Mercken, L., and Octave, J.-N. (1996) Biochem. Biophys. Res. Commun. 218, 89 –96 26. Xu, H., Sweeney, D., Wang, R., Thinakaran, G., Lo, A. C. Y., Sisodia, S., Greengard, P., and Gandy, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3748 –3752 27. Tsunoda, S., Sierralta, J., Sun, Y., Bodner, R., Suzuki, E., Becker, A., Socolich, M., and Zuker, C. S. (1997) Nature 388, 243–249 28. Ermekova, K. S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T., and Sudol, M. (1997) J. Biol. Chem. 272, 32869 –32877 29. Busciglio, J., Gabuzda, D. H., Matsudaira, P., and Yankner, B. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2092–2096 30. Wertkin, A. M., Turner, R. S., Pleasure, S. J., Golde, T. E., Younkin, S. G., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9513–9517 31. Turner, R. S., Suzuki, N., Chyung, A. S. C., Younkin, S. G., and Lee, V. M.-Y. (1996) J. Biol. Chem. 271, 8966 – 8970 32. Tienari, P. J., Ida, N., Ikonen, E., Simons, M., Weidemann, A., Multhaup, G., Masters, C. L., Dotti, C. G., and Beyreuther, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4125– 4130 33. Cook, D. G., Forman, M. S., Sung, J. C., Leight, S., Kolson, D. L., Iwatsubo, T., Lee, V. M.-Y., and Doms, R. W. (1997) Nat. Med. 3, 1021–1023 34. Cook, D. G., Sung, J. C., Golde, T. E., Felsenstein, K. M., Wojczyk, B. S., Tanzi, R. E., Trojanowski, J. Q., Lee, V. M.-Y., and Doms, R. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9223–9228 35. Kovacs, D. M., Fausett, H. J., Page, K. J., Kim, T.-W., Moir, R. D., Merriam, D. E., Hollister, R. D., Hallmark, O. G., Mancini, R., Felsenstein, K. M., Hyman, B. T., Tanzi, R. E., and Wasco, W. (1996) Nat. Med. 2, 224 –229 36. Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181–190 37. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T. D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., and Younkin, S. (1996) Nat. Med. 2, 864 – 870 38. Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., Guido, T., Hagopian, S., Johnson-Wood, K., Khan, K., Lee, M., Leibowitz, P., Lieberburg, I., Little, S., Masliah, E., McConlogue, L., Montoya-Zavala, M., Mucke, L., Paganini, L., Penniman, E., Power, M., Schenk, D., Seubert, P., Snyder, B., Soriano, F., Tan, H., Vitale, J., Wadsworth, S., Wolozin, B., and Zhao, J. (1995) Nature 373, 523–527 39. Hsiao, K., Chapman, P. Nilsen S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F., and Cole, G. (1996) Science 274, 99 –102 40. Sturchler-Pierrat, C., Abramowski, D., Duke, M., Wiederholt, K.-H., Mistl, C., Rothacher, S., Lederman, B., Burki, K., Frey, P., Paganetti, P. A., Waridel, C., Calhoun, M. E., Jucker, M., Probst, A., Staufenbiel, M., and Sommer, B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13287–13292