BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production

Transmembrane proteins BRI2 and amyloid precursor protein (APP) co-localize with amyloid beta (Abeta) lesions in sporadic Alzheimer disease and mutations in both precursor proteins are linked to early-onset familial cases of cerebral amyloidosis associated with dementia and/or cerebral hemorrhage. A specific interaction between BRI2 and APP was unveiled by immunoprecipitation experiments using transfected and non-transfected cells. The use of deletion mutants further revealed that stretches 648-719 of APP751 and 46-106 of BRI2, both inclusive of the full transmembrane domains, are sufficient for the interaction. Removal of most of the APP and BRI2 extracellular domains without affecting the interaction implies that both proteins interact when are expressed on the same cell membrane (cis) rather than on adjacent cells (trans). The presence of BRI2 had a modulatory effect on APP processing, specifically increasing the levels of cellular APP as well as beta-secretase-generated COOH-terminal fragments while decreasing the levels of alpha-secretase-generated COOH-terminal fragments as well as the secretion of total APP and Abeta peptides. Determining the precise molecular pathways affected by the specific binding between APP and BRI2 could result in the identification of common therapeutic targets for these sporadic and familial neurodegenerative disorders.     

Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.     

Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes

Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Abeta production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Abeta. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Abeta.     

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.     

Retention of the Alzheimer's amyloid precursor fragment C99 in the endoplasmic reticulum prevents formation of amyloid beta-peptide

gamma-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's beta-amyloid precursor protein (APP), resulting in the release of amyloid beta-peptide (Abeta). The molecular identity of gamma-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that gamma-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Abeta. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Abeta(42)/Abeta(40) ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of gamma-secretase cleavage. Biogenesis of both Abeta(40) and Abeta(42) was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for gamma-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as gamma-secretase. The results suggest that presenilins may acquire the characteristics of gamma-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.     

Effects of apolipoprotein E (apoE) isoforms, beta-amyloid (Abeta) and apoE/Abeta complexes on protein kinase C-alpha (PKC-alpha) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts

We investigated the effects of different apolipoprotein E (apoE) isoforms, Abeta (1-42), and apoE/Abeta complexes on PKC-alpha translocation and APP processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Treatment of cells with either 10 nM apoE3 or apoE4, 10 microM Abeta (1-42), or apoE/Abeta complexes induced significant translocation of PKC-alpha in both cell types. Effects were seen using both human recombinant apoE and apoE loaded into beta-very low density lipoprotein (beta-VLDL) particles. Time course (5-24 h) studies of APP processing revealed that some conditions induced transient or moderate increases in the secretion of proteins detected by 22C11. In contrast, the secretion of alpha-secretase cleaved APP was either not modified or transiently decreased, as determined by immunoblotting with the antibody 6E10. These results suggest that apoE, Abeta (1-42) and apoE/Abeta complexes can modulate PKC activity but do not have major consequences for APP processing. These effects could contribute to the reported PKC alterations seen in AD. However, it is unlikely that the contribution of different apoE isoforms to AD pathology occurs via effects on APP processing.     

RNA interference-mediated silencing of X11alpha and X11beta attenuates amyloid beta-protein levels via differential effects on beta-amyloid precursor protein processing

Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.     

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.     

Sequences from the low density lipoprotein receptor-related protein (LRP) cytoplasmic domain enhance amyloid beta protein production via the beta-secretase pathway without altering amyloid precursor protein/LRP nuclear signaling

Increasing evidence suggests that the low density lipoprotein receptor-related protein (LRP) affects the processing of amyloid precursor protein (APP) and amyloid beta (Abeta) protein production as well as mediates the clearance of Abeta from the brain. Recent studies indicate that the cytoplasmic domain of LRP is critical for this modulation of APP processing requiring perhaps a complex between APP, the adaptor protein FE65, and LRP. In this study, we expressed a small LRP domain consisting of the C-terminal 97 amino acids of the cytoplasmic domain, or LRP-soluble tail (LRP-ST), in CHO cells to test the hypothesis that the APP.LRP complex can be disrupted. We anticipated that LRP-ST would inhibit the normal interaction between LRP and APP and therefore perturb APP processing to resemble a LRP-deficient state. Surprisingly, CHO cells expressing LRP-ST demonstrated an increase in both sAPP secretion and Abeta production compared with control CHO cells in a manner reminiscent of the cellular effects of the APP "Swedish mutation." The increase in sAPP secretion consisted mainly of sAPPbeta, consistent with the increase in Abeta release. Further, this effect is LRP-independent, as the same alterations remained when LRP-ST was expressed in LRP-deficient cells but not when the construct was membrane-anchored. Finally, deletion experiments suggested that the last 50 amino acid residues of LRP-ST contain the important domain for altering APP processing and Abeta production. These observations indicate that there are cellular pathways that may suppress Abeta generation but that can be altered to facilitate Abeta production.     

Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abeta1-42.

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.     

The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein

We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.     

Regulation of amyloid precursor protein processing by presenilin 1 (PS1) and PS2 in PS1 knockout cells

The presenilin 1 (PS1) and PS2 proteins are thought to play roles in processing of amyloid precursor protein (APP), but the nature of this role is not fully understood. Recent studies have shown that PS1 is necessary for cleavage of APP at the gamma-secretase site. We now show that PS1 and PS2 participate in other aspects of APP processing. Fibroblasts generated from PS1 knockout mice have increased levels of the APP cleavage products, secreted APP (APPs), and APP C-terminal fragments, but lower secretion of APPs and Abeta. We have also observed that loss of PS1 prevents protein kinase C or extracellular regulated kinase from increasing production of the APP cleavage products, APPs, and APP C-terminal fragments. Transfection of PS1 -/- cells with PS1 restores the responsiveness of APP processing to protein kinase C and extracellular regulated kinase. This suggests that the changes in APP processing in PS1 -/- cells result strictly from the absence of PS1. Transfection of PS1 -/- cells with PS2 is also able to correct the deficits in APP secretion, which suggests that the PS2 also has the ability to regulate APP processing. Finally, transfection of the truncated PS2 construct, Alg3, into cells lacking PS1 increases APP C-terminal fragments. This suggests that Alg3 can interfere with the processing of APP by PS2. These data point to roles for both PS1 and PS2 in regulating APP processing and suggest that the role of these proteins also includes coupling APP to signal transduction pathways.     

Role of endoplasmic reticulum, endosomal-lysosomal compartments, and microtubules in amyloid precursor protein metabolism of human neurons

A wide interest in amyloid precursor protein (APP) metabolism stems from the fact that increased amounts of amyloid beta peptide (Abeta), arising through proteolytic processing of APP, likely play a significant role in Alzheimer's disease. As Alzheimer's disease pathology is limited almost exclusively to the human species, we established human primary neuron cultures to address the possibility of distinctive APP processing in human CNS neurons. In the present study, we investigate the role of organelles and protein trafficking in APP metabolism. Using brefeldin A, we failed to detect APP processing into Abeta in the endoplasmic reticulum. Monensin and the lysomotropic agents, NH4Cl and chloroquine, revealed a bypass pH-dependent secretory pathway in a compartment between the endoplasmic reticulum and the medial Golgi, resulting in the secretion of full-length APP. Colchicine treatment resulting in the loss of neurites inhibited processing of APP through the secretory, but not the endosomal-lysosomal, pathway of APP metabolism. The serine protease inhibitor, leupeptin, indicates a role for lysosomes in APP, Abeta, and APP C-terminal fragment turnover. These results demonstrate that the regulation of APP metabolism in human neurons differs considerably from those reported in rodent CNS primary neuron cultures or continuously dividing cell types.     

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.     

Expression, purification and characterization of a synthetic gene encoding human amyloid beta (Abeta1-42) in Escherichia coli

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Existing evidence indicates that abnormal processing and extracellular deposition of the longer form of the amyloid peptide Abeta(1-42), a proteolytic derivative of the amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Active immunization with Abeta(1-42) has been shown to decrease brain beta deposition and improve cognitive performance in mouse models of AD. In the present study, we sought to express the synthetic gene encoding AB in Escherichia coli to enable rapid production of the antigen and its purification. The synthetic gene has been constructed from six oligonucleotides by employing overlapping PCR strategy and expressed in E. coli using the T7 promoter system. The recombinant peptide has been purified to homogeneity by a single step Ni+2 affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-Abeta(1-42) sera confirms that the corresponding linear B-cell epitopic sequences are available for immunorecognition in the recombinant peptide. This methodology enables rapid, continuous production and purification in bulk amounts of human Abeta sequence by employing bacterial expression system     

Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B

Alzheimer amyloid precursor protein (APP) is the precursor for the Abeta peptide involved in pathogenesis of Alzheimer's disease. The soluble ectodomain fragment of APP (sAPP) functions as a growth factor for epithelial cells, suggesting an important function for APP outside neuronal tissue. Previous studies have shown that in polarized epithelial cells, APP is targeted to the basolateral domain. Tyr653 within the cytoplasmic tail of APP mediates the basolateral targeting of APP, but the sorting machinery that binds to this residue has largely remained unknown. In this study, we analyzed the role of adaptor complexes in the polarized sorting of APP. We show that the medium subunit mu1B of the epithelia-specific adaptor protein (AP)-1B binds onto the cytoplasmic tail of APP in a Tyr653-dependent way. Moreover, ectopic expression of mu1B in cells lacking AP-1B resulted in correction of apical missorting of wild-type but not Tyr653Ala APP. Basolateral secretion of sAPP was found to be independent of Tyr653. We propose a model for polarized targeting of APP according to which sorting of APP to basolateral domain is dependent on binding of AP-1B on Tyr653 in basolateral endosomes. This model is in accordance with the current understanding of sorting mechanisms mediating polarized targeting of membrane proteins.     

BACE is degraded via the lysosomal pathway

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.     

Presenilin-1, nicastrin,amyloid precursor protein,and gamma-secretase activity are co-localized in the lysosomal membrane

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.     

Fyn modulation of Dab1 effects on amyloid precursor protein and ApoE receptor 2 processing

Dab1 is an intracellular adaptor protein that interacts with amyloid precursor protein (APP) and apoE receptor 2 (apoEr2), increases their levels on the cell surface, and increases their cleavage by alpha-secretases. To investigate the mechanism underlying these alterations in processing and trafficking of APP and apoEr2, we examined the effect of Fyn, an Src family-tyrosine kinase known to interact with and phosphorylate Dab1. Co-immunoprecipitation, co-immunostaining, and fluorescence lifetime imaging demonstrated an association between Fyn and APP. Fyn induced phosphorylation of APP at Tyr-757 of the (757)YENPTY(762) motif and increased cell surface expression of APP. Overexpression of Fyn alone did not alter levels of sAPPalpha or cytoplasmic C-terminal fragments, although it significantly decreased production of Abeta. However, in the presence of Dab1, Fyn significantly increased sAPPalpha and C-terminal fragments. Fyn-induced APP phosphorylation and cell surface levels of APP were potentiated in the presence of Dab1. Fyn also induced phosphorylation of apoEr2 and increased its cell surface levels and, in the presence of Dab1, affected processing of its C-terminal fragment. In vivo studies showed that sAPPalpha was decreased in the Fyn knock-out, supporting a role for Fyn in APP processing. These data demonstrate that Fyn, due in part to its effects on Dab1, regulates the phosphorylation, trafficking, and processing of APP and apoEr2.