T668 phosphorylation is up-regulated in AD brains
To investigate P-APP in normal and disease brain samples, we generated a rabbit polyclonal antibody against a phospho-T668 containing peptide (αP-T668). We carefully characterized these antibodies and confirmed that the αP-T668 specifically recognized P-APP in vitro and in rat brain lysates (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1
). Next, we used αP-T668 for immunohistochemistry to determine the T668 phosphorylation status of APP in AD brain samples (case information in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1
). The overall staining patterns of P-APP in age-matched control and AD brains differed markedly (, A–E). Intense staining was observed in AD hippocampal pyramidal neurons (). Pre-adsorption of the antibody with the P-T668 containing peptide, but not the unphosphorylated peptide, completely eliminated the staining (Fig. S2, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200301115/DC1
). Alkaline phosphatase pretreatment of AD hippocampal sections abolished αP-T688 staining (Fig. S2, Da and b). Both mammalian APP homologues, APLP1 and APLP2 contain a homologous phosphorylated threonine in their cytoplasmic domains. Pre-adsorption of αP-T668 with phosphorylated APLP1 or APLP2 peptides containing these sites did not affect the staining pattern produced by this antibody (Fig. S2, Ea–d) making it unlikely that the αP-T668 staining in AD brains was due to phosphorylation of APLP1 or APLP2. Together, these results indicate that the αP-T668 specifically labels structures containing T668 phosphorylated APP in AD brains.
Figure 1. T668 phosphorylated APP is elevated in human AD brains. (A) Age-matched control hippocampal section stained with αP-T668. (B) AD hippocampal section stained with αP-T668. Strong immunoreactivity is detected in pyramidal neurons and some (more ...)
We have examined the localization of P-APP in a total of 24 AD and 11 nondemented age-matched control cases. In general, two structures that displayed strong P-T668 staining were large, vesicular bodies within the cell soma of hippocampal neurons ( D) and dystrophic neurites associated with amyloid plaques (, E–I). We also determined the distribution of P-APP in the brains of 18 mo-old APPsw Tg mice and found that αP-T668 labeled dystrophic neurites that are closely associated with amyloid plaques (Fig. S2, F–I). However, no vesicular staining in the cell body is observed. Thus, in the mouse model for amyloid plaques, P-APP is only present in one of the two compartments where it is enriched in AD.
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 ( 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 ( 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 ( 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.
Figure 2. The CTFs of T668 phosphorylated APP is elevated in AD brains. (A) Western blots of brain lysates from control and AD hippocampal tissues probed with αP-T668 or an antibody against the COOH terminus of APP (C1/6.1). (B) Schematic diagram of peptides (more ...)
To determine phosphorylation sites of APP CTFs, in addition to T668, we immunoprecipitated APP from AD hippocampal lysates using αP-T668, resolved and trypsin digested the APP CTFs in a SDS-PAGE, and analyzed the tryptic fragments using MALDI-TOF mass spectrometry (MS). A summary of recovered peptides and a representative MS spectrum of tryptic digests are shown in (B and C). The recovered peptides spanned the entire APP βCTF region. The presence of peptides 596–612, 596–624, 602–612 and 602–624 indicated the existence of βCTF (C99). However, as trypsin also cleaves at the α-secretase site, we cannot rule out the presence of αCTF or other species of APP CTFs in the MS samples. Treatment of samples with alkaline phosphatase before MS analysis significantly reduced the intensities of the phospho-peptide signals and resulted in new ion peaks appearing at m/z positions representing dephosphorylated peptides, confirming that these peptides were indeed phosphorylated (unpublished data).
Interestingly, we found that peptides spanning residues 650–672 and 651–672 contained three phosphates per peptide. Peptides 652–676 and 673–695 contained four phosphates per peptide ( C). The sequence composition of the peptide ion with m/z 3123 (peptide 652–676) 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 673–695 with four phosphates suggested that all potential sites (S675, Y682, T686, and Y687) were phosphorylated ( 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 651–672 and 650–672) 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 673–695, 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 ( 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 αP-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-APP–positive vesicles could be labeled by the endosome markers Rab4 ( A), Rab5 ( B), and EEA1 ( C), but not by the lysosome markers cathepsin D ( 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.
Figure 3. Subcellular localization of P-APP in AD brain sections. (A–D) P-APP is localized to endocytic compartments but not lysosomes in AD brains. T668 phosphorylated APP colocalizes with endocytic vesicle markers: (A) Rab4, (B) Rab5, and (C) EEA1, but (more ...)
We also examined the presence of APP processing enzymes in the P-APP–positive vesicles. Interestingly, the staining of β-secretase BACE1 was robust in the P-APP–positive neurons. Furthermore, BACE1 displayed extensive colocalization with P-APP ( E). A second BACE1 antibody gave rise to similar staining pattern in AD brain sections (unpublished data). Antibodies to PS1 did not specifically label the P-APP–positive vesicles in AD brain sections (unpublished data). These data suggest that P-APP colocalizes with BACE1 in enlarged endosomes in AD brains.
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 (). Interestingly, P-APP showed substantial colocalization with the early endosome marker Rab5 in the growth cones (). Modest overlap between P-APP and EEA1 (early endosome marker; E), Rab4 (recycling endosome marker; F), Rab7 (late endosome marker; G) or adaptin-γ (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 () 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 ( C). P-APP and PS1 also showed little colocalization in the growth cones ( D).
Figure 4. Subcellular localization of P-APP in cultured primary cortical neurons. Double immunofluorescence staining of rat primary cortical neurons. (A and B) Costaining of P-APP and regular APP (C1/6.1) in the (A) soma and (B) growth cone. (C and D) Colocalization (more ...)
Figure 5. T668 phosphorylated APP colocalizes with BACE1 in primary cortical neurons. (A and B) Costaining of P-APP and BACE1 in the growth cones. (C) Costaining of regular APP (C1/6.1) and BACE1 in the growth cone. (D) Costaining of P-APP and Presenilin 1(PS1) (more ...)
We further performed immunoisolation experiments to characterize P-APP, APP, and BACE1 containing vesicles ( E). We found that Rab5 was present in P-APP, APP, and BACE1 immunoisolates. Previous studies have shown that APP is present in the TGN (Annaert et al., 1999
). However, we did not detect GM130 in any of the immunoisolates. In addition, the levels of BACE1 in P-APP immunoisolates were higher than those in APP immunoisolates, indicating that T668 phosphorylated APP preferentially colocalized with BACE1. Together, these experiments suggest that P-APP is in the same subcellular compartment as the APP processing enzyme BACE1, in both AD brains and cultured neurons.
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 ( A). Western blot analysis using the APP COOH-terminal antibody showed that full-length APP had a broad distribution between fractions 8–20. Full-length P-APP displayed a more restricted profile between fractions 8–16. We also detected P-APP signal in the bottom of the gradient (fractions 21–23). 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 9–13, whereas APP CTFs were present between fractions 8–16. The APP CTFs detected by the αP-T668 displayed a higher molecular size than those detected by the APP COOH-terminal antibody, indicating that αP-T668 might preferentially label the β-secretase product(s) of APP.
Figure 6. T668 phosphorylated APP cofractionates with BACE1 and endosome markers. (A) Adult mouse brain homogenates were fractionated through an iodixanol step gradient. Western blot analysis showed the distribution of P-APP, APP, Rab5 (early endosome marker), (more ...)
Organelle markers revealed that Rab5 displayed a broad distribution, which overlapped with P-APP. Lysosomes, as identified by cathepsin D, also displayed a broad distribution, which was shifted to the right of the gradient. The Golgi apparatus (GM130) and ER (Bip) segregated to the bottom of the gradient. Digitalization and plotting of the Western blot signals of fractions 1–20 revealed that full-length APP, P-APP and BACE1 largely cosegregated in fractions 8–13 of iodixanol gradient with the APP signal extended to the heavier fractions of the gradient ( B).
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 ( 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 ( 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 β-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 β-secretase products of APP, as markers for identifying different CTF species (, 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 βCTFs and those with faster mobility than C89 as αCTFs. In these brain lysates, αCTFs were much more abundant than βCTFs (, bottom). Increasing amounts of αP-T668 efficiently immunoprecipitated βCTFs, as recognized by the APP COOH-terminal antibody, in a dose-dependent manner ( A, top). A corresponding dose-dependent decrease in βCTFs was observed in the supernatant of these immunoprecipitates ( A, bottom). On the other hand, the level of αCTFs in the supernatants only decreased slightly. When 10 μg of αP-T668 was used, ~60% of βCTFs were depleted, whereas only <10% of αCTFs were depleted from the brain lysates ( C). As a control, we used the APP COOH-terminal antibody to immunoprecipitate APP CTFs from mouse brain lysates. This antibody efficiently removed both αCTFs and βCTFs from the lysates (). These observations suggest that the β-secretase products of APP are preferentially phosphorylated on T668 in vivo and raise the possibility that T668 phosphorylation may facilitate APP cleavage by BACE1.
Figure 7. βCTFs are preferentially phosphorylated on T668. (A) Increasing amounts of αP-T668 was used to immunoprecipitate APP from lysates prepared from a 6-mo-old mouse brain. Western blot analysis using a pan APP antibody (C1/6.1) showed that (more ...)
Effects of T668 phosphorylation on Aβ generation in primary cortical neurons
To elucidate whether T668 phosphorylation plays a role in Aβ generation, we assessed Aβ levels from the cultured media of neurons treated with the Cdk inhibitors roscovitine or butyrolactone. Because the levels of secreted Aβ from endogenous APP was too low to be detected, we used recombinant HSV to express wild-type APP in rat cortical neurons. Both roscovitine and butyrolactone treatments caused substantial decreases in T668 phosphorylation ( B). Interestingly, levels of secreted Aβ 1-40 and 1-42 were significantly reduced by these inhibitors in a dose-dependent manner ( A). The reduction in Aβ secretion was not due to a decline in cell viability as determined by the MTT assay (unpublished data).
Figure 8. Reduced Aβ generation by APP T668 to alanine mutant and T668 kinase inhibitors in primary cortical neurons. (A) Inhibiting T668 phosphorylation by Cdk inhibitors leads to a decrease in Aβ secretion. Rat primary cortical neurons were treated (more ...)
Butyrolactone and roscovitine are general Cdk inhibitors. Because Cdks phosphorylate many substrates, it is possible that the reduction in Aβ secretion is an indirect effect resulting from reduced phosphorylation of proteins other than APP. To directly test the hypothesis that T668 phosphorylation is involved in the metabolism of APP, we compared Aβ levels generated from neurons expressing wild-type versus T668A mutant APP. Rat cortical neurons 2 d in vitro were infected with HSV expressing either wild-type or the T668A mutant APP. The Aβ levels in the culture media were determined 20 h after infection. D showed that the expression levels of wild-type and T668A mutant APP were comparable in these cultures. However, Aβ 1-40 and 1-42 levels in neuronal cultures expressing the T668A mutant APP were significantly reduced compared with those expressing wild-type APP ( C). These results suggest that phosphorylation of T668 regulates APP processing and Aβ generation.