Article |
Address correspondence to Li-Huei Tsai, Dept. of Pathology, Harvard Medical School and Howard Hughes Medical Institute, 200 Longwood Ave., Boston, MA 02115. Tel.: (617) 432-1053. Fax: (617) 432-3975. email: li-huei_tsai{at}hms.harvard.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Alzheimer's disease; amyloid precursor protein; BACE1; endosomes; Aß
The online version of this paper contains supplemental material.
Abbreviations used in this paper: Aß, amyloid-ß peptide; AD, Alzheimer's disease; APLP, APP-like protein; APP, amyloid precursor protein; CTF, COOH-terminal fragment; HSV, herpes simplex virus; MS, mass spectrometry; P-APP, APP phosphorylated on threonine 668; PNS, postnuclear supernatant; PS, presenilin.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pharmacological and biochemical studies have shown that ß- and -secretases are aspartyl proteases (Wolfe et al., 1999; Yan et al., 1999). ß-Secretase (BACE 1) and its homologue BACE2 are type I integral membrane glycoproteins (Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). BACE1 constitutes the primary ß-secretase activity in the brain and is primarily localized within the Golgi and endosomal compartments where the acidic pH is optimal for secretase activities. To date, there is no known genetic linkage between mutations in BACE genes and AD. However, BACE1 proteins and activities have been found to be increased in AD brain regions affected by amyloid deposition, suggesting its role in AD pathogenesis (Fukumoto et al., 2002; Holsinger et al., 2002; Yang et al., 2003).
Presenilin proteins (PS1 and PS2) are integral components of the -secretase directly responsible for the activity of
-secretase (Steiner and Haass, 2000). Importantly, missense mutations within PS genes are associated with familial forms of AD. These mutations result in increased production of Aß42, accelerating the formation of senile plaques (Hardy and Crook, 2001). PS proteins are mostly localized in the endoplasmic reticulum, Golgi apparatus (Kovacs et al., 1996; De Strooper et al., 1997), and endosomes (Lah and Levey, 2000).
Aß has been shown to be generated in both the secretory and endocytic pathways of transfected cell lines and cultured neurons (Selkoe et al., 1996; Perez et al., 1999; Nixon et al., 2000). It is thought that Aß can first be generated in the TGN. Remaining unprocessed APP is then transported to the cell surface where it is either cleaved by the nonamyloidogenic -secretase or reinternalized into the endosomes, where Aß generation has also been found. Disturbances in neuronal endocytic pathway are among the earliest known intracellular changes occurring in sporadic AD (Nixon et al., 2000). In hippocampal neurons of these AD patients, endosomes are abnormally enlarged; a change that may precede clinical symptoms, appearing before substantial Aß accumulation. As such, abnormalities associated with the endocytic pathway have been postulated to play a significant role in amyloidogenesis (Cataldo et al., 2000).
How APP is targeted to the subcellular compartments containing its processing enzymes remains to be elucidated. Subcellular APP trafficking may be regulated by its phosphorylation. APP contains eight potential phosphorylation sites in its cytoplasmic domain. Low level phosphorylation of T654 and S655 (human APP695 isoform numbering) has been found in rat brain (Oishi et al., 1997). These two amino acids are part of the 653YTSI656 motif, which acts as an endocytic and/or basolateral sorting signal, YXXI, in MDCK cells (Lai et al., 1998; Zheng et al., 1998). Tyrosine kinases, TrkA and c-Abl, have been shown to phosphorylate Y682 in vitro (Tarr et al., 2002). The 682YENPTY687 motif is a canonical endocytic signal for membrane-associated receptors and mutations of Y682, N684, or P685 have been shown to inhibit the internalization of APP and decrease the generation of secreted sAPP and Aß (Perez et al., 1999; Steinhilb et al., 2002).
Threonine 668 (T668) in the cytoplasmic domain of APP is also phosphorylated in vivo by a number of protein kinases, including GSK3ß, SAPK1b/JNK3, Cdc2, and Cdk5 (Suzuki et al., 1994; Aplin et al., 1996; Iijima et al., 2000; Standen et al., 2001). Many of these kinases are associated with neurotoxicity and implicated in neurodegenerative diseases. A solution NMR study suggests that T668 phosphorylation results in significant conformational change that may affect the interactions of APP with its binding partners (Ramelot and Nicholson, 2001). As such, this phosphorylation may serve to regulate the intracellular trafficking and metabolism of APP.
Here, we provide evidence that T668 phosphorylated APP is significantly increased in AD brains. APP phosphorylated on threonine 668 (P-APP) and BACE1 colocalize in enlarged endosomes of AD hippocampal neurons and cultured primary cortical neurons. P-APP and BACE1 from mouse brain homogenates cofractionate in iodixanol gradients. Mutation of T668 to alanine (T668A) decreases this cofractionation. Furthermore, the COOH-terminal fragments of APP generated by BACE1 cleavage, ßCTFs (C99/C89), are preferentially phosphorylated over the -secretase cleavage product,
CTF (C83). Accordingly, Aß generation is significantly reduced by mutation of T668 to alanine or by treatment using T668 kinase inhibitors. Together, these observations suggest that T668 phosphorylation may facilitate ß-secretase cleavage of APP and generation of Aß peptides.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
T668 phosphorylated APP accumulates in neurons positive for phospho-Tau
Double immunostaining of P-APP and phospho-Tau (AT8) revealed a high coincidence of staining within neurons from AD (Fig. 1 J). Among 1,373 neurons surveyed that were positive for P-T668, AT8 or both, 81% were double positive, 3% were only positive for P-T668 and 16% were only positive for AT8. Of the neurons positive for AT8 only, most represented extracellular tangles that were remnants of degenerated neurons. This observation indicates that afflicted neurons in AD exhibit increased levels of P-APP. Although P-APP and phospho-Tau were present in the same neurons, they exhibited distinct subcellular localization; P-APP was present in vesicular compartments, whereas phospho-Tau was associated with filamentous structures (Fig. 1 K).
Phosphorylation of COOH-terminal fragments of APP in AD brains
To further investigate the phosphorylation of T668 in AD, we performed Western blot analysis on hippocampal tissues from 14 AD and 10 age-matched control brains (Fig. 2 A; case information in Table S2, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1). No significant difference in levels of P-T668 on full-length APP was observed between AD and control brains. However, we found P-T668 on the APP CTFs to be considerably increased in 8 of 14 AD brains.
|
Interestingly, we found that peptides spanning residues 650672 and 651672 contained three phosphates per peptide. Peptides 652676 and 673695 contained four phosphates per peptide (Fig. 2 C). The sequence composition of the peptide ion with m/z 3123 (peptide 652676) was selected and analyzed by postsource decay MS. We found that Y653, S655, and T668 were phosphorylated, based on the presence of corresponding y-ions and a phospho-tyrosine immonium ion (unpublished data). Furthermore, the recovery of peptide 673695 with four phosphates suggested that all potential sites (S675, Y682, T686, and Y687) were phosphorylated (Fig. 2 C). These results indicate that in AD brains, APP CTFs contain at least seven different sites that can be phosphorylated, including T668. As a control, we isolated APP CTFs from neuroblastoma CAD cells overexpressing human APP using an APP COOH-terminal antibody. We found two phospho-peptides (peptides 651672 and 650672) containing three phosphates per peptide, indicating that three out of four potential sites (Y653, T654, S655, and T668) were phosphorylated. We did not detect any signal representing peptide 673695, which contained four potential phosphorylation sites (S675, Y682, T686, and Y687; see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1). This result showed that analogous phospho-peptides of APP CTFs detected in AD brain samples were also present in recombinant APP expressing CAD cells. Moreover, as many potential sites were not phosphorylated in APP expressing cells, it supports the notion that the cytoplasmic domain of APP is hyperphosphorylated in AD. Because the phosphorylated residues include serines, threonines and tyrosines, this observation suggests that multiple protein kinases are involved in APP phosphorylation in AD.
T668 phosphorylated APP is enriched in endocytic compartments and colocalized with BACE1 in AD brains
The large vesicular structures positive for P-APP have not been described previously (Fig. 1 D). To determine the nature of this structure, we performed double immunofluorescence staining on AD hippocampal sections with a large panel of organelle markers. As P-T668 recognizes both full-length APP and APP CTFs, immunostaining using this antibody represents localization of both forms of APP phosphorylated on T668. We found that P-APPpositive vesicles could be labeled by the endosome markers Rab4 (Fig. 3 A), Rab5 (Fig. 3 B), and EEA1 (Fig. 3 C), but not by the lysosome markers cathepsin D (Fig. 3 D) or cathepsin B (Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1), the synaptic vesicle marker SV2 (not depicted), the Golgi markers GM130 or MannII (not depicted), or the ER markers Bip/Grp78 (not depicted) or GP96 (Fig. S4 B). These costaining results indicate that P-APP is enriched in the endocytic compartments of AD hippocampal neurons.
|
Colocalization of T668 phosphorylated APP and BACE1 in primary neurons
To further determine the subcellular distribution of P-APP and its physiological relationship with BACE1, we performed double immunostaining on normal rat primary cortical neurons. In these neurons, P-APP signal appeared punctate in the soma and growth cones with a distribution pattern somewhat distinct from that of regular APP (Fig. 4, A and B). Interestingly, P-APP showed substantial colocalization with the early endosome marker Rab5 in the growth cones (Fig. 4, C and D). Modest overlap between P-APP and EEA1 (early endosome marker; Fig. 4 E), Rab4 (recycling endosome marker; Fig. 4 F), Rab7 (late endosome marker; Fig. 4 G) or adaptin- (TGN marker; Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1) was observed. There was little colocalization between GM130 (cis-Golgi marker; Fig. S5 B) and P-APP. Interestingly, P-APP and BACE1 displayed extensive colocalization in the growth cones of young neurons (Fig. 5, A and B) and showed partial colocalization in neurites of 10-d-old cultured neurons (Fig. S5, C and D). Only limited colocalization of regular APP and BACE1 was observed (Fig. 5 C). P-APP and PS1 also showed little colocalization in the growth cones (Fig. 5 D).
|
|
T668 phosphorylated APP cofractionates with BACE1 and endosome markers in an iodixanol step gradient
Next, we performed biochemical fractionations to gain additional insight into the subcellular localization of P-APP. Organelles in an adult wild-type mouse brain were separated through an iodixanol step gradient (Fig. 6 A). Western blot analysis using the APP COOH-terminal antibody showed that full-length APP had a broad distribution between fractions 820. Full-length P-APP displayed a more restricted profile between fractions 816. We also detected P-APP signal in the bottom of the gradient (fractions 2123). As the gradient was bottom loaded, this likely represented unsegregated lysates, or possibly, the presence of immature, less glycosylated P-APP in the early secretory pathway. P-APP CTFs had a very discrete distribution spanning fractions 913, whereas APP CTFs were present between fractions 816. The APP CTFs detected by the P-T668 displayed a higher molecular size than those detected by the APP COOH-terminal antibody, indicating that
P-T668 might preferentially label the ß-secretase product(s) of APP.
|
To assess the significance of T668 phosphorylation in APP subcellular localization, we introduced wild-type and T668A mutant APP into primary cortical neurons using recombinant herpes simplex virus (HSV). 20 h after infection, cell homogenates were fractionated through iodixanol step gradient and the distribution of APP was analyzed. Interestingly, wild-type APP exhibited more extensive cosegregation with BACE1 than the T668A mutant APP, which was shifted to the heavier fractions of the gradient (Fig. 6 C; immunoblots in Fig. S6 A, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1). T668 of APP can be phosphorylated by multiple kinases including Cdc2 and Cdk5. The activities of these Cdks can be inhibited by pharmacological reagents roscovitine and butyrolactone. We analyzed APP distribution from roscovitine treated cortical neurons by fractionation using iodixanol gradient. Similarly, we found that APP distribution shifted to heavier membrane fractions after roscovitine treatment (Fig. 6 D; immunoblots in Fig. S6 B). This observation indicates that T668 phosphorylation plays a role in the intracellular trafficking of APP.
APP CTFs generated by ß-secretase are preferentially phosphorylated on T668
To further determine the species of APP CTFs that is phosphorylated on T668 in vivo, we performed immunodepletion experiments. We used CTFs derived from CAD cells overexpressing C99, a ß-secretase products of APP, as markers for identifying different CTF species (Fig. 7, A and B, lane 1). We found that CTFs from mouse brain lysates generally showed slower mobility than those from C99 overexpressing CAD cells, possibly due to differences in the stoichiometry of phosphorylation. As such, we assigned those CTFs with slower mobility than C99 from CAD cells as ßCTFs and those with faster mobility than C89 as CTFs. In these brain lysates,
CTFs were much more abundant than ßCTFs (Fig. 7, A and B, bottom). Increasing amounts of
P-T668 efficiently immunoprecipitated ßCTFs, as recognized by the APP COOH-terminal antibody, in a dose-dependent manner (Fig. 7 A, top). A corresponding dose-dependent decrease in ßCTFs was observed in the supernatant of these immunoprecipitates (Fig. 7 A, bottom). On the other hand, the level of
CTFs in the supernatants only decreased slightly. When 10 µg of
P-T668 was used,
60% of ßCTFs were depleted, whereas only <10% of
CTFs were depleted from the brain lysates (Fig. 7 C). As a control, we used the APP COOH-terminal antibody to immunoprecipitate APP CTFs from mouse brain lysates. This antibody efficiently removed both
CTFs and ßCTFs from the lysates (Fig. 7, B and C). These observations suggest that the ß-secretase products of APP are preferentially phosphorylated on T668 in vivo and raise the possibility that T668 phosphorylation may facilitate APP cleavage by BACE1.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
P-APP and BACE1 signals are enriched in enlarged endosomes in AD brains
It has been recently reported that the expression levels and activities of BACE1 are up-regulated in AD brains, especially in neurons of temporal cortex and hippocampus (Fukumoto et al., 2002; Holsinger et al., 2002). Interestingly, we found that BACE1 signal is enriched in the enlarged endosomes of hippocampal pyramidal neurons in AD brain sections. Furthermore, BACE1 colocalizes with P-APP in these enlarged endosomes. Analysis of AD brain lysates reveals that phosphorylation of APP CTFs on T668 is up-regulated. In addition, we also found that the ßCTFs are preferentially phosphorylated on T668. Together, these observations suggest that P-APP is preferentially cleaved by the increased BACE1 in AD brain, possibly in the endosomes. Enhanced endocytic activity is one of the earliest known neuropathological alternations in sporadic Alzheimer's disease. Abnormalities associated with the endocytic pathway have been proposed to play a role in Aß generation (Cataldo et al., 2000; Nixon et al., 2000). Recent studies by Grbovic et al. (2003) demonstrated that Aß generation could be up-regulated by enhanced endocytic activity as a result of overexpression of Rab5. As such, the drastic up-regulation and accumulation of BACE1 and P-APP in the endocytic vesicles implies the significance of the enlarged endosomes in contributing to the altered processing of APP in AD.
P-APP is preferentially associated with BACE1 in the endocytic pathway in primary cortical neurons
Endocytic pathway is one of the major sites where Aß is normally generated (Nordstedt et al., 1993; Koo and Squazzo, 1994). The presence of APP in the Rab5-positive endocytic compartments has been shown previously in rat brain nerve terminals (Ikin et al., 1996). Here, we found that P-APP is present in Rab5 labeled endocytic vesicles and exhibits extensive colocalization with BACE1 in growth cones of young cortical neurons. This observation is further supported by the coimmunoisolation of Rab5 with P-APP and BACE1-positive vesicles. In addition, P-APP and BACE1 cosegregate in the iodixanol gradient. In the gradient, the fractionation profiles of P-APP and BACE1 largely overlap with Rab5. As for older neurons, the colocalization of P-APP and BACE1 is less prominent. Although endosomes are highly enriched in the growth cones of young neurons, endosomes of mature neurons are concentrated in branch region of axons and presynaptic nerve terminals (Overly and Hollenbeck, 1996). Studies by Sabo et al. showed that APP is localized in Rab5 containing synaptic organelles in mature neurons (Sabo et al., 2003). It is possible that P-APP and BACE1 colocalize in specific loci of neurites, such as areas where synapses form in mature neurons.
Unlike the staining pattern of AD brains, we detected modest colocalization of P-APP with the early endosome marker EEA1, in primary cortical neurons. In the early endocytic pathway, Rab5 regulates both clathrin-coated vesicle-mediated transport from the plasma membrane to the early endosomes, as well as homotypic early endosome fusion, whereas EEA1 medicates homotypic early endosome fusion. Our findings suggest that in normal conditions, P-APP is localized in Rab5-positive endocytic vesicles that do not actively undergo homotypic endosome fusion. In addition, we did not observe extensive colocalization of P-APP with recycling endosome marker Rab4 in primary cortical neurons. It is likely that the colocalization of P-APP and EEA1/Rab4 in AD brains is due to abnormalities in endocytic pathway associated with the disease state. We detected little colocalization of P-APP with the TGN marker adaptin- and the cis-Golgi marker GM130. These observations suggest that P-APP is preferentially exposed to BACE1 in the Rab5-positive endosomes. Although BACE1 cleaves APP in both the secretory pathway and endocytic compartments, our results suggest that T668 phosphorylated APP is likely the target of BACE1 in the Rab5-positive endocytic compartments in both normal and disease conditions.
T668 phosphorylation regulates APP processing
A contribution of T668 phosphorylation on APP processing is underscored by the significantly reduced Aß 1-40 and 1-42 secretion from neurons treated with T668 kinase inhibitors or neurons expressing the T668A mutant APP. In normal mouse brain lysates, the CTF is much more abundant than the ßCTFs. Interestingly,
P-T668 preferentially recognizes ßCTFs over
CTF and efficiently depletes ßCTFs from the brain lysates. This observation strongly suggests that P-APP is more likely to be cleaved by the ß-secretase. Whether T668 phosphorylation of APP directly or indirectly impacts on BACE1 cleavage remains to be determined.
We found that the T668A mutant produces 50% less Aß compared with the wild-type APP in cultured neurons. This observation indicates that in the absence of T668 phosphorylation, Aß is still generated, albeit less efficiently. Therefore, it is less likely that T668 phosphorylation directly impacts on APP cleavage. Rather, T668 phosphorylation is likely to be involved in the intracellular sorting and trafficking of APP, which in turn impacts on the proteolytic cleavage of APP. This view is supported by the observation that the T668A mutant is mislocalized as evident by its distribution in the heavier fractions of iodixanol gradient.
Previous studies by Ando et al. (2001) reported that T668 glutamate mutant of APP (T668E) did not affect Aß generation in HEK293 cells. Unlike the T668E mutant, expression of the T668A mutant causes mislocalization of APP and a decrease in Aß generation. This, together with the observation that Cdk inhibitors roscovitine and butyrolactone elicit the same effect as T668A mutant, strongly argues that T668 phosphorylation of APP plays a role in regulating trafficking of APP and Aß generation.
From solution NMR studies, it has been predicted that phosphorylation of T668 may result in a dramatic conformational change of the APP cytoplasmic tail, which is likely to affect the interaction of APP with its binding partners. Indeed, Ando and colleagues showed that T668 phosphorylation weakens the interaction of APP with Fe65 (Ando et al., 2001). However, the impact of the interactions of APP with Fe65 on the metabolism of APP is less clear. Guenette et al. (1996) and Sabo et al. (1999) reported that expressing Fe65 in MDCK and H4 neuroglioma cells lead to increased accumulation of APP on the cell surface and increased secretion of Aß. On the contrary, overexpression of Fe65 resulted in reduced Aß secretion in HEK293 cells (Ando et al., 2001). As such, the effect of Fe65 appears to be cell-type specific. It is possible that disruption of the APPFe65 interaction through phosphorylation of T668 represents one of the mechanisms underlying increased generation of Aß.
When considered together, our findings that T668 phosphorylation facilitates APP and BACE1 colocalization and that inhibition of T668 phosphorylation decreases Aß generation suggest that T668 phosphorylation of APP is a molecular mechanism regulating its cleavage by BACE1. As BACE1 cleavage is the essential step for Aß generation, understanding how proteolysis by BACE1 is regulated will help elucidate the intricate intracellular signaling network regulating the generation of Aß.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
Anti-APP (C1/6.1) was a gift from R.A. Nixon and P.M. Mathews (both from Nathan Kline Institute, Orangeburg, NY). 4G8 was obtained from Senetek. AT8 was obtained from Innogenetics. Anti-BACE1 was obtained from Oncogene Research Products and Calbiochem. Anti-Rab5 and anti-Bip were obtained from StressGen Biotechnologies. Anti-Rab4, anti-Rab7, and anticathepsin D were obtained from Santa Cruz Biotechnology, Inc. Antiadaptin- and anti-GM130 were obtained from BD Biosciences. Anti-PS1 mAbs and pAbs were obtained from CHEMICON International, Inc. and a gift from D. Selkoe (Harvard Medical School). Anti-SV2 and anticathepsin ß were gifts from K.M. Buckley and H.L. Ploegh, respectively (both from Harvard Medical School).
Generation of P-T668
The phospho-epitopespecific APP antibody was generated against a synthetic peptide antigen corresponding to APP phosphorylated at the T668 residue: VDAAVpTPEERHC, where pT denotes phosphothreonine (Tufts Peptide Synthesis Core Facility). The peptide was conjugated to KLH with sulfo-MBS (Pierce Chemical Co.) and injected into NZW rabbits (Covance Research Products Inc.). The antiserum was first adsorbed against a corresponding nonphosphorylated peptide, VDAAVTPEERHC, and purified with the phospho-peptide coupled to a SulfoLink coupling column (Pierce Chemical Co.).
Cell line and neuronal cell culture
Catecholaminergic cell lines (CAD cells) were cultured in DME supplemented with 10% FBS and L-glutamine in a humidified 5% CO2 incubator.
Primary cultures of embryonic rat cortical neurons were prepared as described previously (Niethammer et al., 2000). In brief, dissociated embryonic neurons from E18 Sprague-Dawley pregnant rats were plated onto poly-D-lysine/laminincoated 24-well plates or coverslips and maintained in neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), L-glutamine (Sigma-Aldrich) and 1% penicillin-streptomycin sulfate.
Generation of recombinant HSV
Wild-type and T668A APP coding sequence were subcloned into a replication-defective HSV vector pHSVPrpUC. The resultant recombinant plasmid was packaged into virus particles in the packaging line 22 using the protocol described previously (Lim et al., 1996). The virus was then purified on a sucrose gradient, pelleted, and resuspended in 10% sucrose and the titer of the recombinant virus was determined.
Immunohistochemistry
AD and control tissues were obtained from the autopsy service at Brigham and Women's Hospital. Neuropathological diagnosis of AD was confirmed according to the criteria of Khachaturian (1985). Blocks of AD or control hippocampi were fixed (148 h) in 10% neutral buffered formalin. After fixation, the brain tissue was dehydrated and embedded in paraffin. 20 micrometer serial sections were cut, dried, and baked at 60°C for 1 h. Serial sections were immunostained using the avidin-biotin HFP/DAB method (Vector Laboratories) or by immunofluorescence. Details of the immunostaining protocol have been described previously (Lemere et al., 1996). All images were captured using an inverted microscope (Nikon) linked to a DeltaVision deconvolution imaging system (Applied Biosystems). For some immunofluorescence staining, the primary antibody was directly labeled with Oregon green using the FluoReporter Oregon green 488 protein labeling kit (Molecular Probes) according to manufacturer's specifications.
For the peptide preadsorption experiment, antibodies were mixed with peptides at a 1:100 molar ratio and incubated overnight at 4°C before being used for staining.
Immunocytochemistry
Primary cortical neurons from E18 rat embryos were cultured at a density of 105 cells/well in 24-well plates. 2 d after plating, neurons were fixed in 4% PFA for 30 min, blocked, and permeabilized in 10% normal goat serum and 0.1% Triton in PBS for 20 min. Permeabilized neurons were incubated with primary antibodies for 1 h at room temperature, and subsequently incubated with Oregon green or Texas redconjugated secondary antibodies (Molecular Probes). Images were captured using an inverted microscope (Nikon) linked to a DeltaVision deconvolution imaging system (Applied Biosystems).
Immunodepletion
Immunodepletion was performed by lysing the mouse brain in RIPA buffer (50 mM Tris, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and protease and phosphatase inhibitors [1 mM PMSF, 1 µg/ml aprotinin and leupeptin, and 20 mM ß-glycerol phosphate]) using a Dounce homogenizer. The lysates were centrifuged at 13,000 rpm for 15 min at 4°C. 300 µg of lysates were incubated with indicated amounts of antibodies and 30 µl of 50% slurry of protein ASepharose (Amersham Biosciences) at 4°C for 2.5 h. The immunoprecipitates were washed three times with RIPA buffer, resuspended in Laemmli sample buffer, and analyzed by Western blot analysis.
Immunoisolation
Adult mouse brain was homogenized in 2 ml of immunoisolation buffer (250 mM sucrose, 20 mM Hepes, pH 7.3, 5 mM MgCl2,, and protease and phosphatase inhibitors) by 20 strokes in a Dounce homogenizer followed by an additional 20 strokes in a 1-ml syringe fitted with 21-gauge needle. Lysates were centrifuged at 3,000 rpm for 15 min. The postnuclear supernatant (PNS) was collected and centrifuged at 27,000 rpm for 30 min to precipitate vesicles. The resulting pellet was resuspended in immunoisolation buffer that did not contain sucrose. Equal amounts of vesicle suspension were incubated at 4°C overnight with indicated antibody that had been preconjugated to M-280 Tosylactivated Dynabeads (Dynal) according to the manufacturer's instructions. Immunoisolates were washed three times with immunoisolation buffer containing 0.1% BSA, resuspended in Laemmli sample buffer, and subjected to Western blot analysis.
Mass spectrometric analysis
Blocks of AD hippocampi (gifts from M. Frosch, Massachusetts General Hospital, Boston, MA) were lysed in RIPA buffer using a Dounce homogenizer. T668 phosphorylated APP were isolated using a P-T668 column according to the manufacturer's instructions (Amersham Biosciences). Isolated proteins were resolved using SDS-PAGE. Protein bands containing APP-CTFs were cut and subjected to S-carbamidomethylation and in-gel trypsin digestion (Stensballe and Jensen, 2001). Peptide digests were extracted in 25 mM ammonium bicarbonate, pH 8.8, containing 25% (vol/vol) dimethyl foramide and further purified using ZipTipC18 (Millipore). Purified peptides were mixed with
cyano-4-hydroxycinnamic acid matrix and analyzed by Voyager DE-STR mass spectrometer (PerSeptive Biosystems). Postsource decay experiments were performed according to the protocols provided the manufacturer. Tables of mass/charge (m/z) of trypsinized APP peptides were generated using Protein Prospector (http://prospector.ucsf.edu/). The tolerance of the difference between experimental and theoretical m/z values in comparison was constrained under 300 ppm.
Iodixonal step gradient
Half of a 1-mo-old adult mouse brain was homogenized in 1 ml of homogenization buffer (HB: 250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, and protease and phosphatase inhibitors) by 20 strokes in a Dounce homogenizer followed by an additional 20 strokes in a 1-ml syringe fitted with 21-gauge needle. Lysates were centrifuged at 3,000 rpm for 15 min to generate the PNS. The PNS was then adjusted to 25% OptiPrep (Nycomed/Axis-Shield PoC.) with 50% OptiPrep in HB. The resulting mixture, 2 ml in 25% OptiPrep, was placed at the bottom of an ultracentrifuge tube (14 x 89 mm) and was overlaid successively with 1 ml each of 20, 18.5, 16.5, 14.5, 12.5, 10.5, 8.5, 6.5, and 5% OptiPrep in cold HB. The gradients were centrifuged for 20 h at 27,000 rpm at 4°C in a rotor (model SW41; Beckman Coulter). 500µl fractions were collected from the top of the ultracentrifuge tubes and analyzed by Western blot analysis. P-APP, APP, and BACE levels in each fraction were digitized by a Luminescent Image Analyzer (Fujifilm) and expressed as a percentage of the sum of all of the fractions.
Aß measurement by ELISA assay
T668 kinase inhibitor.
Primary cortical neurons were cultured from E18 rat embryos at a density of 4 x 105 cells/well in 24-well plates. 2 d after plating, neurons were infected with recombinant HSV expressing wild-type APP for 16 h. Subsequently, 70% of culture media was replaced with fresh medium and neurons were treated with indicated concentration of Roscovitine or Butyrolactone. 8 h after inhibitor treatment, culture media was collected and subjected to sandwich ELISA assay according to the manufacturer's specifications (Biosource International).
T668A mutant.
Primary cortical neurons from E18 rat embryos were cultured at a density of 2 x 105 cells/well in 24-well plates. 2 d after plating, 70% of culture media was replaced with fresh medium. Neurons were subsequently infected with equal titer of HSV expressing either wild-type or T668A APP. 20 h after infection, culture media was collected and subjected to sandwich ELISA assay. We observed most dramatic effect on Aß generation 24 h after infection. By 48 h after infection, the effect is less pronounced. Data were analyzed by t test using Prism (GraphPad). Differences were considered significant at P < 0.05.
Online supplemental material
The supplemental material (Figs. S1S6 and Tables S1 and S2) is available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1. Fig. S1 shows characterization of P-T668. Fig. S2 shows additional controls for
P-T668 staining of AD brain sections and that
P-T668 labels dystrophic neurites closely associated with amyloid plaques in APPswe Tg mice. Fig. S3 shows a partial MALDI-TOF MS spectrum of APP CTFs from CAD cells overexpressing human APP. Figs. S4 and S5 show costaining of P-APP and organelle markers in AD brain sections and in rat primary cortical neurons. Fig S6 shows distribution of wild-type, T668A mutant APP, and BACE1 from cultured cortical neurons in iodixanol step gradient. Tables S1 and S2 show case information of human brains analyzed by immunohistochemistry and Western blot analysis.
![]() |
Acknowledgments |
---|
This project was partially supported by MetLife Foundation Award made to L.-H. Tsai. L.-H. Tsai is an Associate Investigator of the Howard Hughes Medical Institute.
Submitted: 28 January 2003
Accepted: 11 August 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ando, K., K.I. Iijima, J.I. Elliott, Y. Kirino, and T. Suzuki. 2001. Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of beta-amyloid. J. Biol. Chem. 276:4035340361.
Annaert, W.G., L. Levesque, K. Craessaerts, I. Dierinck, G. Snellings, D. Westaway, P.S. George-Hyslop, B. Cordell, P. Fraser, and B. De Strooper. 1999. Presenilin 1 controls -secretase processing of amyloid precursor protein in pre-Golgi compartments of hippocampal neurons. J. Cell Biol. 147:277294.
Aplin, A.E., G.M. Gibb, J.S. Jacobsen, J.M. Gallo, and B.H. Anderton. 1996. In vitro phosphorylation of the cytoplasmic domain of the amyloid precursor protein by glycogen synthase kinase-3beta. J. Neurochem. 67:699707.[Medline]
Cataldo, A.M., C.M. Peterhoff, J.C. Troncoso, T. Gomez-Isla, B.T. Hyman, and R.A. Nixon. 2000. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am. J. Pathol. 157:277286.
De Strooper, B., and W. Annaert. 2000. Proteolytic processing and cell biological functions of the amyloid precursor protein. J. Cell Sci. 113:18571870.
De Strooper, B., M. Beullens, B. Contreras, L. Levesque, K. Craessaerts, B. Cordell, D. Moechars, M. Bollen, P. Fraser, P.S. George-Hyslop, and F. Van Leuven. 1997. Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins. J. Biol. Chem. 272:35903598.
Fukumoto, H., B.S. Cheung, B.T. Hyman, and M.C. Irizarry. 2002. Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch. Neurol. 59:13811389.
Grbovic, O.M., P.M. Mathews, Y. Jiang, S.D. Schmidt, R. Dinakar, N.B. Summers-Terio, B.P. Ceresa, R.A. Nixon, and A.M. Cataldo. 2003. Rab5-stimulated up-regulation of the endocytic pathway increases intracellular levels of beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production. J. Biol. Chem. 278:3126131268.
Guenette, S.Y., J. Chen, P.D. Jondro, and R.E. Tanzi. 1996. Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein. Proc. Natl. Acad. Sci. USA. 93:1083210837.
Hardy, J., and R. Crook. 2001. Presenilin mutations line up along transmembrane alpha-helices. Neurosci. Lett. 306:203205.[CrossRef][Medline]
Holsinger, R.M., C.A. McLean, K. Beyreuther, C.L. Masters, and G. Evin. 2002. Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Ann. Neurol. 51:783786.[CrossRef][Medline]
Iijima, K., K. Ando, S. Takeda, Y. Satoh, T. Seki, S. Itohara, P. Greengard, Y. Kirino, A.C. Nairn, and T. Suzuki. 2000. Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5. J. Neurochem. 75:10851091.[CrossRef][Medline]
Ikin, A.F., W.G. Annaert, K. Takei, P. De Camilli, R. Jahn, P. Greengard, and J.D. Buxbaum. 1996. Alzheimer amyloid protein precursor is localized in nerve terminal preparations to Rab5-containing vesicular organelles distinct from those implicated in the synaptic vesicle pathway. J. Biol. Chem. 271:3178331786.
Khachaturian, Z.S. 1985. Diagnosis of Alzheimer's disease. Arch. Neurol. 42:10971105.[CrossRef][Medline]
Koo, E.H., and S.L. Squazzo. 1994. Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269:1738617389.
Kovacs, D.M., H.J. Fausett, K.J. Page, T.W. Kim, R.D. Moir, D.E. Merriam, R.D. Hollister, O.G. Hallmark, R. Mancini, K.M. Felsenstein, et al. 1996. Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells. Nat. Med. 2:224229.[Medline]
Lah, J.J., and A.I. Levey. 2000. Endogenous presenilin-1 targets to endocytic rather than biosynthetic compartments. Mol. Cell. Neurosci. 16:111126.[CrossRef][Medline]
Lai, A., A. Gibson, C.R. Hopkins, and I.S. Trowbridge. 1998. Signal-dependent trafficking of beta-amyloid precursor protein-transferrin receptor chimeras in madin-darby canine kidney cells. J. Biol. Chem. 273:37323739.
Lemere, C.A., J.K. Blusztajn, H. Yamaguchi, T. Wisniewski, T.C. Saido, and D.J. Selkoe. 1996. Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation. Neurobiol. Dis. 3:1632.[CrossRef][Medline]
Lim, F., D. Hartley, P. Starr, P. Lang, S. Song, L. Yu, Y. Wang, and A.I. Geller. 1996. Generation of high-titer defective HSV-1 vectors using an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells. Biotechniques. 20:460469.[Medline]
Niethammer, M., D.S. Smith, R. Ayala, J. Peng, J. Ko, M.S. Lee, M. Morabito, and L.H. Tsai. 2000. NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein. Neuron. 28:697711.[Medline]
Nixon, R.A., A.M. Cataldo, and P.M. Mathews. 2000. The endosomal-lysosomal system of neurons in Alzheimer's disease pathogenesis: a review. Neurochem. Res. 25:11611172.[CrossRef][Medline]
Nordstedt, C., G.L. Caporaso, J. Thyberg, S.E. Gandy, and P. Greengard. 1993. Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells. J. Biol. Chem. 268:608612.
Oishi, M., A.C. Nairn, A.J. Czernik, G.S. Lim, T. Isohara, S.E. Gandy, P. Greengard, and T. Suzuki. 1997. The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells. Mol. Med. 3:111123.[Medline]
Overly, C.C., and P.J. Hollenbeck. 1996. Dynamic organization of endocytic pathways in axons of cultured sympathetic neurons. J. Neurosci. 16:60566064.
Perez, R.G., S. Soriano, J.D. Hayes, B. Ostaszewski, W. Xia, D.J. Selkoe, X. Chen, G.B. Stokin, and E.H. Koo. 1999. Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J. Biol. Chem. 274:1885118856.
Price, D.L., S.S. Sisodia, and S.E. Gandy. 1995. Amyloid beta amyloidosis in Alzheimer's disease. Curr. Opin. Neurol. 8:268274.[Medline]
Ramelot, T.A., and L.K. Nicholson. 2001. Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR. J. Mol. Biol. 307:871884.[CrossRef][Medline]
Sabo, S.L., L.M. Lanier, A.F. Ikin, O. Khorkova, S. Sahasrabudhe, P. Greengard, and J.D. Buxbaum. 1999. Regulation of beta-amyloid secretion by FE65, an amyloid protein precursor-binding protein. J. Biol. Chem. 274:79527957.
Sabo, S.L., A.F. Ikin, J.D. Buxbaum, and P. Greengard. 2003. The amyloid precursor protein and its regulatory protein, FE65, in growth cones and synapses in vitro and in vivo. J. Neurosci. 23:54075415.
Selkoe, D.J., T. Yamazaki, M. Citron, M.B. Podlisny, E.H. Koo, D.B. Teplow, and C. Haass. 1996. The role of APP processing and trafficking pathways in the formation of amyloid beta-protein. Ann. NY Acad. Sci. 777:5764.[Abstract]
Sinha, S., J.P. Anderson, R. Barbour, G.S. Basi, R. Caccavello, D. Davis, M. Doan, H.F. Dovey, N. Frigon, J. Hong, et al. 1999. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature. 402:537540.[CrossRef][Medline]
Standen, C.L., J. Brownlees, A.J. Grierson, S. Kesavapany, K.F. Lau, D.M. McLoughlin, and C.C. Miller. 2001. Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3). J. Neurochem. 76:316320.[CrossRef][Medline]
Steiner, H., and C. Haass. 2000. Intramembrane proteolysis by presenilins. Nat. Rev. Mol. Cell Biol. 1:217224.[CrossRef][Medline]
Steinhilb, M.L., R.S. Turner, and J.R. Gaut. 2002. ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways. J. Neurochem. 80:10191028.[CrossRef][Medline]
Stensballe, A., and O.N. Jensen. 2001. Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface. Proteomics. 1:955966.[CrossRef][Medline]
Suzuki, T., M. Oishi, D.R. Marshak, A.J. Czernik, A.C. Nairn, and P. Greengard. 1994. Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein. EMBO J. 13:11141122.[Abstract]
Tarr, P.E., R. Roncarati, G. Pelicci, P.G. Pelicci, and L. D'Adamio. 2002. Tyrosine phosphorylation of the beta-amyloid precursor protein cytoplasmic tail promotes interaction with Shc. J. Biol. Chem. 277:1679816804.
Vassar, R., B.D. Bennett, S. Babu-Khan, S. Kahn, E.A. Mendiaz, P. Denis, D.B. Teplow, S. Ross, P. Amarante, R. Loeloff, et al. 1999. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 286:735741.
Wolfe, M.S., W. Xia, B.L. Ostaszewski, T.S. Diehl, W.T. Kimberly, and D.J. Selkoe. 1999. Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature. 398:513517.[CrossRef][Medline]
Yan, R., M.J. Bienkowski, M.E. Shuck, H. Miao, M.C. Tory, A.M. Pauley, J.R. Brashier, N.C. Stratman, W.R. Mathews, A.E. Buhl, et al. 1999. Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature. 402:533537.[CrossRef][Medline]
Yang, L.B., K. Lindholm, R. Yan, M. Citron, W. Xia, X.L. Yang, T. Beach, L. Sue, P. Wong, D. Price, et al. 2003. Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat. Med. 9:34.[CrossRef][Medline]
Zheng, P., J. Eastman, S. Vande Pol, and S.W. Pimplikar. 1998. PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein. Proc. Natl. Acad. Sci. USA. 95:1474514750.
Related Article