ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711

APP669-711 is proteolytically generated from various cells under physiological conditions

Although γ-secretase-mediated C-terminal variations of Aβ have been extensively analyzed, the N-terminal variations of secreted Aβ have not been investigated to date. Importantly, several analyses of cell-based models and genetically modified animals harboring the APP mutant carrying the Swedish mutation (APPswe) located at the (−1st) and (−2nd) positions of Aβ, which significantly increases the β-secretase-mediated cleavage at the 1st position, to induce the overproduction of Aβ have been performed [18, 19]. However, the profiles of secreted Aβ from endogenous APP and overexpressed APPswe are distinct in mouse neuroblastoma Neuro2a cells [20]. We previously demonstrated using IP-MALDI-MS and the 6E10 antibody that human neuroblastoma BE(2)-C cells secrete endogenous APP669-711 as well as Aβ [12]. We further analyzed the conditioned media from several human-derived cell lines. We found that APP669-711 was secreted from A549 adenocarcinoma cells as well as HEK293A cells expressing wild-type APP (APPwt), but not CCF-STTG1 astrocytoma, H4 neuroglioma, nor naïve HEK293A cells (Fig. 1C), suggesting that APP669-711 is produced from only some cultured cell lines under physiological conditions. To investigate the mechanism of the generation of APP669-711, we analyzed the effects of several inhibitors that affect APP metabolism in BE(2)-C cells (Fig. 1D, E). The γ-secretase inhibitor DAPT abolished the production of APP669-711. In contrast, treatment with the specific and potent BACE1 inhibitor MBSI increased APP669-711 production. Notably, GM6001, a pan metalloprotease inhibitor, partially inhibited APP669-711 production, although the ADAM10/17-specific inhibitor INCB3619 did not affect APP669-711 levels. These data suggested that an unknown metalloprotease-mediated cleavage at the APP669 site (a.k.a. (−3rd) position in Aβ) is involved in the generation of APP669-711.

Fig. 1: Detection of APP669-711 in the conditioned medium of cultured cell lines.
figure 1

A IP-MALD-MS spectrum of the 6E10 antibody-precipitated endogenous Aβ peptide variants in the conditioned medium from BE(2)-C cells. Various Aβ peptides, which are diminished by either MBSI or DAPT treatment, were identified. B The identified Aβ peptides secreted in A. OxAβ1-40 contained an oxidized methionine at the Aβ35th residue. C Comparison of the level of endogenous APP669-711 in the conditioned medium of human-derived cultured cells (n = 4–6, mean ± s.e.m. Tukey test). D Pharmacological effects of secretase inhibitors on APP metabolism in BE(2)-C cells. Asterisks indicate non-specific bands. E Pharmacological effects of secretase inhibitors on the production of APP669-711 from BE(2)-C cells (n = 4 or 5, mean ± s.e.m. Tukey test).

In the Aβ production pathway, APP is cleaved by BACE1 at the 1st position of Aβ to generate c99, which is a direct substrate for γ-secretase. According to the proteolytic model above, the APP669-site cleavage of APP results in the production of the C-terminal stub c102, which is c99 with 3 extra N-terminal amino acid residues. To detect endogenous c102, we generated N-terminal end-specific anti-c102 antibodies (anti-c102#1 for immunoblotting, and anti-c102#3 for immunohistochemistry). The specificity of the antibodies was confirmed by immunoblotting of HEK293A cell lysates expressing recombinant c99 or c102, the latter being a sequence with 3 extra amino acid residues between the signal peptide and Aβ sequence in the c99 expression vector (Fig. 2A). By IP using the anti-c102 antibody, we confirmed the appearance of endogenous c102 in the lysates of A549 cells upon DAPT treatment (Fig. 2B, C). Furthermore, cotreatment of GM6001 reduced c102 levels in A549 cell lysates, supporting our notion that APP669-site cleavage of APP is mediated by an unknown metalloprotease. To further confirm that c102 is a direct substrate for APP669-711, we analyzed the conditioned medium of HEK293A cells expressing APPwt, c99, and c102 by IP-MALDI-MS (Fig. 2D, E). As expected, we detected the Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, Aβ1-42, and APP669-711 (Aβ(-3)-40) in the media of APPwt-expressing cells. From recombinant c99, various Aβ species starting at the 1st position were identified. In contrast, the conditioned medium of cells expressing c102 contained various Aβ species starting at APP669 position (i.e., APP669-708 (Aβ(-3)-37), APP669-709 (Aβ(-3)-38), APP669-710 (Aβ(-3)-39), APP669-711 (Aβ(-3)-40), APP669-713 (Aβ(-3)-42)). DAPT treatment completely abolished the production of all Aβ species derived from APPwt and c102 (Fig. 2F, G). These data strongly indicate that APP669-711 is also proteolytically produced from APP by sequential cleavage by a yet-unknown metalloprotease at the APP669 site, and γ-secretase within the transmembrane domain.

Fig. 2: Processing of c102, a direct substrate for APP669-711.
figure 2

A Immunoblot analysis of HEK293A cells expressing APP, c102, and c99. Note that the N-terminal end-specific antibodies anti-c102 and 82E1 detected c102 and c99, respectively. B Detection of endogenous c102 in the lysate of A549 cells by IP using an anti-c102 antibody. Cells were treated with DAPT to increase c102 levels. C Changes in c102 levels by GM6001 or MBSI in A549 cells co-treated with DAPT (n = 3, mean ± s.e.m. Tukey test). D IP-MALD-MS spectrum of the 6E10 antibody-precipitated Aβ peptides in the conditioned medium from HEK293 cells expressing APP, c102, or c99. I.S. internal standard. E Levels of Aβ peptides secreted from HEK293A cells transfected with APP, c102, or c99 (n = 4 or 5 or 4, mean ± s.e.m.). F Effects of DAPT on the production of Aβ peptides of APP-expressing HEK293A cells. G Effects of DAPT on the production of Aβ peptides of c102-expressing HEK293A cells.

We then comprehensively analyzed the endogenous Aβ species secreted from mouse neuroblastoma Neuro2a cells by IP-MALDI-MS. For IP, instead of the 6E10 antibody, which is a human Aβ-specific antibody, we utilized the 4G8 antibody targeting the common sequence in the middle of human and murine Aβ (Fig. 3A, B). We successfully detected 34 types of Aβ-associated peptides in the cultured medium (Fig. 3C). As observed in human-derived cultured cells, GM6001 treatment of neuro2A cells significantly reduced the production of endogenous APP669-711, whereas MBSI did not affect APP669-711 levels. Intriguingly, treatment with MBSI, but not GM6001, abolished the production of Aβ species starting at the 1st, 2nd, and 11th positions, suggesting that BACE1 activity is specifically required for the proteolysis of APP at these sites. In contrast, the production of Aβ species starting at the APP669 and Aβ 12th positions was significantly reduced by GM6001, but not MBSI. Furthermore, we analyzed the effects of the Tissue inhibitor of metalloproteinase 3 (TIMP3), which is an endogenous potent metalloprotease inhibitor implicated in AD pathogenesis [21, 22], on secreted APP669-711 levels from Neuro2a cells (Fig. 3D). Notably, TIMP3 treatment completely inhibited the production of endogenous APP669-711, whereas secreted Aβ levels were unaltered. A comparison of absolute levels of Aβ1-40 and APP669-711 indicated that the processing ratio of BACE1 and ADAMTS4 on APP is 1:0.15. We then estimated the proportion of APP undergoing processing by BACE1 and α-secretase by end-specific ELISAs for sAPPβ and sAPPα, respectively, as endogenous Aβ17-40 was not detected in IP-MALDI-MS spectra. A comparison of absolute levels of sAPPβ and sAPPα was 1:4.1. Thus, we speculate that the proportion of endogenous APP substrates undergoing processing by the BACE1, α-secretase, and ADAMTS4 is 1:4.1:0.15. These data support our notion that the metalloprotease-mediated proteolytic production pathway for APP669-711 is independent of the Aβ-generating pathway, and is conserved in murine cells.

Fig. 3: Metalloprotease-dependent production of APP669-711 from Neuro2a cells.
figure 3

A IP-MALD-MS spectrum of the 4G8 antibody-precipitated endogenous Aβ peptides in the conditioned medium from Neuro2a cells. Various Aβ peptides were identified. I.S. internal standard. B The pharmacological effect of secretase inhibitors on the production of APP669-711 from Neuro2a cells (n = 3, mean ± s.e.m. Tukey test). C Comparison of the effects of GM6001 and MBSI on the production of endogenous Aβ peptides in Neuro2a cells. D Effects of TIMP3 treatment of on the APP669-711 and Aβ production from Neuro2a cells (n = 3, mean ± s.e.m. Tukey test. N.S. not significant).

Identification of ADAMTS4 as an APP669 site-cleaving enzyme

A comprehensive analysis of various Aβ peptides from GM6001-treated cells suggests that the same metalloprotease is involved in cleaving APP669 and the Aβ 12th positions of Aβ. Recently, it was reported that the Aβ 12th position is cleaved by the secreted metalloprotease ADAMTS4 [23]. ADAMTS4 is identified as aggrecanase-1, which is a major aggrecan-degrading enzyme and is implicated in the development of osteoarthritis [24, 25]. The proteolytic activity of ADAMTS4 is inhibited by TIMP3 [26, 27]. The recognition motif for ADAMTS4 (i.e., E-[AFVLMY]-X[0,1]-[RK]-X[2,3]-[ST]-[VYIFWMLA]) contains Glu at the P1 position and partially corresponds to the (−4th) position in the human Aβ sequence, in addition to the Aβ 3rd and Aβ 11th positions [28]. Of note, the Aβ 3rd position in the murine Aβ sequence does not match with the motif, as the Aβ 5th position is Gly instead of Arg in humans. Walter et al. reported that the overexpression of ADAMTS4 in HEK293 cells expressing APPswe resulted in the overproduction of Aβ4-40 and Aβ12-40 [23]. They also showed that the Aβ peptide starting at the 1st position is also a direct substrate for ADAMTS4. However, they did not investigate the effects of ADAMTS4 on APP669-711 production.

To test whether ADAMTS4 can cleave APP directly at the APP669 site, we first performed the in vitro digestion assay using a novel recombinant APP substrate, APP81 (Fig. 4A). APP81 encodes the APP619-699 sequence tagged with N-terminal FLAG and C-terminal V5-His tags. Coincubation of purified APP81 with recombinant ADAMTS4 resulted in the appearance of low-molecular-weight peptides that were recognized by anti-His tag and anti-Aβ (6E10) antibodies (Fig. 4B). This reaction was specifically inhibited by the addition of metal chelator ethylenediaminetetraacetic acid. We then analyzed the proteolyzed peptides by MALDI-TOF-MS (Fig. 4C, D). All N- and C-terminal fragments generated by APP669, Aβ 4th, and Aβ 12th-site cleavages (FLAG-APP668 and APP669-His; FLAG-Aβ3 and Aβ4-His; and FLAG-Aβ11 and Aβ12-His, respectively) were detected. Moreover, a peptide derived from double digestion by APP669 and Aβ 12th-site cleavages (i.e., APP669-Aβ11) was also identified by MS/MS analysis. These data indicate that ADAMTS4 can directly cleave APP at APP669, the 4th and 12th positions.

Fig. 4: In vitro cleavage assay using recombinant APP81 substrate.
figure 4

A Schematic depiction of the recombinant APP81 substrate and the in vitro assay. B Immunoblot analysis of APP81 (black triangles) and cleaved products (white triangles) generated by recombinant ADAMTS4. Note that the ADAMTS4-mediated cleavage was inhibited by preincubation with EDTA. C MALDI-MS spectrum of the reaction mixture of APP81 and ADAMTS4 (green). Several proteolytic fragments, which were not identified in the substrate only (orange), enzyme only (red), nor EDTA coincubated (blue) samples, were detected. The inset indicates the enlarged spectrum around m/z 6000 to 6600. Asterisks indicate double protonated signals. D Theoretical molecular weights of peptides specifically identified from the reaction mixture, and their annotated sequences are shown.

Next, we analyzed the conditioned medium of HEK293A cells expressing APPwt and ADAMTS4 (Figs. 5A, B and S1). Consistent with a previous report, the level of Aβ4-40 was significantly increased by ADAMTS4 expression [23]. The level of APP669-711 was also slightly, but significantly upregulated by ADAMTS4, and was reduced by treatment with GM6001 or TIMP3. The production of Aβ1-40 and Aβ1-42 was not altered by ADAMTS4 overexpression, GM6001, or TIMP3 treatment. Furthermore, co-expression of c102 with ADAMTS4 did not increase Aβ4-40 levels (Fig. 5C). Notably, the production of APP669-711 from APPwt expressed in HEK293A cells was unaltered by the addition of the conditioned medium of ADAMTS4-expressing HEK293A cells, suggesting that ADAMTS4 cleavage at the APP669 site occurred on APP in the secretory pathway, rather than the cell surface APP (Fig. S2). We then analyzed the effects of the genetic knockout of ADAMTS4 in A549 cells, which secrete endogenous Aβ and APP669-711. IP-MALDI-MS analysis of the conditioned medium of A549 cells using 6E10 antibody revealed that endogenous Aβ4-40 is hardly produced from this cell line unless ADAMTS4 is overexpressed (Fig. S3). Two monoclonal A549 cell lines that harbor deletion/substitution mutations at the ADAMTS4 locus by the CRISPR/Cas9 system were selected (Fig. 5D). Both cell lines showed a 30–40% reduction in secreted APP669-711 levels, whereas Aβ production was unaltered (Fig. 5E). Notably, GM6001 treatment further decreased the production of APP669-711 from ADAMTS4-knockout A549 cells. Finally, we analyzed the effects of the genetic ablation of Adamts4 on the level of endogenous APP669-711 in vivo. Human Aβ and mouse Aβ differ by three amino acids (R5/Y10/H13 in human Aβ are G5/H10/R13 in mouse Aβ), and the antibody APP597 was raised against a synthetic peptide encoding murine Aβ (see Fig. 6B, E) [29]. IP-MALD-MS using the 4G8 antibody or the murine Aβ-specific APP597 antibody enabled us to detect endogenous murine Aβ-associated species, such as Aβ1-40, Aβ1-42, and APP669-711 in wt mouse plasma (Fig. 5F). We then applied this method to the plasma samples obtained from Adamts4−/− mice [30]. Consistent with the data of ADAMTS4-knockout A549 cells, the plasma levels of murine APP669-711 in Adamts4−/− mice were 33% lower than those of wt mouse plasma (Fig. 5G). Collectively, these data strongly suggest that ADAMTS4 is involved in the generation of approximately one-third of the APP669-711 in plasma.

Fig. 5: Effects of ADAMTS4 on APP669-711 production.
figure 5

A Immunoblot analysis of HEK293A cell lysates expressing APP and ADAMTS4. B Levels of Aβ peptides in the conditioned medium of HEK293A cells in A (n = 4 or 5, mean ± s.e.m. Tukey test. N.S. not significant). C Levels of Aβ peptides in the conditioned medium of HEK293A cells expressing c102 and ADAMTS4 (n = 3, mean ± s.e.m. Tukey test). D Target sequence of the ADAMTS4 locus in A549 cells and genomic sequences of the monoclonal A549 cell lines. E Levels of endogenous Aβ peptides in the conditioned medium of A549 cells in D (n = 4 or 5, mean ± s.e.m. Tukey test. N.S. not significant). F IP-MALDI-MS spectrum of the 4G8 or APP597 antibody-precipitated endogenous Aβ peptides in the plasma of wt mice (100 μl for 4G8, 125 μl for APP597). I.S. internal standard (distinct peptides were used for 4G8 and APP597). N non-specific peak. G Levels of endogenous APP669-711 and Aβ1-40 in the plasma of wt and Adamts4 knockout mice (n = 7, mean ± s.e.m. Student’s t test).

Fig. 6: APP669-711 in the Aβ plaque-laden AD model mice.
figure 6

A Effects of the Swedish mutation in APP on the production of APP669-711. Levels of Aβ peptides in the conditioned medium of HEK293A cells expressing APPwt and APPswe are shown. B The specificity of the APP597 antibody against the murine Aβ sequence was analyzed by immunoblot analysis of HEK293A cells expressing human c102, or murine c102. C Immunoblot analysis of brain fractions of aged wt and APP/PS1 mice. TS Tris buffer-soluble fraction, TX 1% Triton-X-soluble fraction, SDS SDS-soluble fraction, FA formic acid-soluble fraction. D Immunohistochemical analysis of the brains of aged APP/PS1 mice. Arrowheads indicate Aβ plaques. E IP-MALDI-MS analysis using APP597 antibody of FA fraction of APP/PS1 mouse (n = 3, 18–22 months old). Wild-type mice (22 months old) were used as a control. Enlarged spectrum in the red box were shown in the inset (black box). F Levels of endogenous mouse Aβ peptides in the plasma of young (2 months old, (−)) and aged (23–25 months old, (+)) APP/PS1 mice. The plasma was analyzed by IP-MALDI-MS using the APP597 antibody (n = 4 or 5, mean ± s.e.m. Student’s t test). G Relative ratio of mouse Aβ peptides in the plasma (n = 4 or 5, mean ± s.e.m. Student’s t test).

Endogenous murine APP669-711 in the brains of Aβ plaque-laden Alzheimer disease model mice

We and others previously reported that the APP669-711 peptide has the ability to form amyloid fibrils [12, 31]. However, the existence of N-terminally elongated Aβ species in amyloid plaques has not been investigated to date. ADAMTS4 is expressed in the central nervous system [24], and our cell-based assay revealed that neuroblastoma cell secretes APP669-711. Furthermore, APP669-711 was detected in the human CSF [32]. Notably, the intergenic variant located near the ADAMTS4 gene has been identified as one of the genetic risk factors for sporadic AD by genome-wide meta analysis [33]. To analyze the pathological effects and the diagnostic value of APP669-711 in the brains of Aβ plaque-laden AD model mice, we analyzed aged mice that express human APPswe and mutant Psen1 (APP/PS1 mice) [34]. Of note, HEK293A cells expressing APPswe produced no APP669-711 carrying the Swedish mutation, suggesting that amino acid substitutions at Aβ (−2nd) and (−1st) sites (i.e., KM670/671NL) abolish the production of APP669-711 from human APP (Fig. 6A). Thus, using the mouse Aβ sequence-specific antibody APP597, we analyzed the effects of the brain deposition of amyloid comprised of human Aβ on the production of APP669-711 from mouse APP, separately from the processing of human APP encoded by the transgene. The specificity of APP597 was confirmed by immunoblot analysis of cell lysate expressing murine c102 (Fig. 6B). For human Aβ, we utilized 82E1 antibody that specifically reacts with N terminus of human Aβ1-x (Fig. 2A). As expected, we detected the deposition of 82E1-positive human Aβ (i.e., human Aβ1-x) in the brain parenchyma and substantial enrichment of human Aβ in the formic acid-soluble fraction. Notably, APP597 reacted with a 4-kDa band corresponding to human Aβ in the formic acid-soluble fraction (Fig. 6C). Furthermore, a similar 4-kDa band was detected by the anti-c102#1 antibody. Consistent with the biochemical analysis, immunohistochemical analysis demonstrated that the plaques in the cortex were positive for APP597 and anti-c102#3 antibodies, suggesting that endogenous murine Aβ peptides including APP669-x peptides formed insoluble aggregates and were deposited in human Aβ plaques. To characterize the deposited endogenous murine Aβ species in the aged APP/PS1 mice, we performed IP-MALDI-MS analysis using APP597. We confirmed that various mouse Aβ peptides, such as Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43 were recovered from the formic acid fraction of APP/PS1 mouse brains. In addition, murine APP669-711 (Aβ(-3)-40), APP669-713 (Aβ(-3)-42), and APP669-714 (Aβ(-3)-43) were detected (Fig. 6E). Finally, we analyzed the level of endogenous murine Aβ species in the plasma of aged APP/PS1 mice, which contained a substantial amount of human Aβ, by IP-MALDI-MS using the APP597 antibody. Comparing the peptide levels of various Aβ species in the plasma of young and aged APP/PS1 mice, we found a significant increase in murine APP669-711/murine Aβ1-42 ratio in the plasma, as observed in the plasma of humans with Aβ plaques in the brain (Fig. 6F, G). However, the plasma murine Aβ1-40/murine Aβ1-42 ratio was unaltered. These results suggest that, in AD model mice with Aβ plaque development, APP669-711 is produced in the brain and deposited together with human Aβ. In addition, the present data also indicate that the levels of mouse Aβ species in plasma are useful as surrogate biomarkers of amyloid deposition in the brains of AD model mice.

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