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Background: We have identified APP669‐711 (a.k.a. Aβ(‐3)‐40) in human plasma using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS), and revealed that the composite biomarker using APP669‐711/Aβ1‐42 ratio correlates with amyloid PET status (Kaneko et al., 2014; Nakamura et al., 2018). We also recently found that A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) is responsible for the APP669‐site cleavage of APP (Matsuzaki et al., Mol Psychiatry 2023). Here, we analyzed the production pathway APP669‐x peptides from the C‐terminal stub c102 of APP, which is c99 with 3 extra N‐terminal amino acid residues (i.e., the cleaved APP fragment at APP669‐site). Method: We established the endo‐specific antibody against the N terminus of APP669‐x peptide, an anti‐c102 antibody. We analyzed the effect of ADAMTS4 on the production of Aβ‐related peptides from the APP‐expressing HEK293 cells using IP‐MALDI‐MS. Result: We successfully detected the endogenous C‐terminal fragment of APP specifically reacted with anti‐c102 antibody in A549 cells. We found that the overexpression of the recombinant c102 resulted in the production of a series of APP669‐x peptides by the γ‐secretase. No Aβ starting at the 1st nor 4th position was generated from c102‐expressing cells irrespective of ADAMTS4 expression. Co‐expression of ADAMTS4 with APP increased the production of both APP669‐711 and Aβ4‐40. However, the addition of extracellular ADAMTS4 to HEK293 cells expressing APP resulted in the increase in the Aβ4‐40, but not APP669‐711 nor Aβ1‐40, suggesting that no further N‐terminal trimming of c102 is occurred within the cells after ADAMTS4‐mediated APP669 cleavage. Conclusion: These results indicated that ADAMTS4 cleaves APP in the secretory pathway to generate c102, which is a direct substrate for the γ‐secretase. [ABSTRACT FROM AUTHOR]

Background: We have previously identified APP669‐711 (a.k.a. Aβ(‐3)‐40) in human plasma using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS) (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014). Our assay has revealed that the composite biomarker, which is a combination of APP669‐711/Aβ1‐42 ratio and Aβ1‐40/ Aβ1‐42 ratio in human plasma, correlates with amyloid PET status (Nakamura et al., Nature 2018). Our previous research using cultured cells has suggested that A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) is involved in APP669‐711 production. Here, we present further evidence on the involvement of ADAMTS4 in the APP669 site‐cleavage. Method: We prepared a recombinant APP substrate, which is APP81 corresponding to APP619‐699 sequence with a V5/His tag at the C‐terminus and a FLAG tag at the N‐terminus (FLAG‐APP81‐V5/His) for in vitro cleavage assay. Recombinant human ADAMTS4 protein and the tagged APP81 were incubated either alone or together at 37°C. The incubated samples were desalted and then analyzed by MALDI‐TOF MS. In vivo evaluation of ADAMTS4, we analyzed plasma samples from Adamts4‐/‐ mice and WT mice using IP‐MALDI‐MS. Result: In vitro cleavage assay, N‐ and C‐terminal fragments cleaved at APP669, Aβ4th, Aβ12th site were detected in the mixture of the recombinant APP81 and ADAMTS4 proteins but not in the sample of substrate or enzyme alone. Furthermore, a peptide derived from double digestion by APP669 and Aβ12th site‐cleavages (i.e., APP669‐Aβ11) was also identified by MS/MS analysis. The analysis of mouse plasma showed that murine APP669‐711 level was decreased in Adamts4‐/‐ mice compared to WT mice. Conclusion: These results indicated that ADAMTS4 is one of the enzymes that can cleave the APP669 site directly and contribute to the generation of APP669‐711 in vivo. [ABSTRACT FROM AUTHOR]

Background: We have previously identified APP669‐711 (a.k.a. Aβ(‐3)‐40) in human plasma using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS) (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014). Our assay has revealed that the composite biomarker, which is a combination of APP669‐711/Aβ1‐42 ratio and Aβ1‐40/ Aβ1‐42 ratio in human plasma, correlates with amyloid PET status (Nakamura et al., Nature 2018). However, the significance of these peptide ratios in an APP/PS1 mouse model of Alzheimer's disease has been unclear. In this study, we investigated the levels of Aβ‐related peptides including APP669‐711 in plasma samples from the model mouse. Method: We prepared plasma samples from young (2 months of age) and old (23–25 months of age) APP/PS1 mice expressing both APP bearing the Swedish mutation and PSEN1 harboring ΔE9 mutation, and cultured supernatants of HEK293 cells. Mouse plasma samples and cultured supernatants were measured by IP‐MALDI‐MS. Result: Human APP669‐711 was detected in cultured supernatant of HEK293 cells overexpressing human wild‐type APP but not human APP with the Swedish mutation. This result indicated that two amino acid substitutions (i.e., Swedish mutation) near the APP669 site inhibited the APP669 cleavage and the production of APP669‐711 from the transgene of APP/PS1 mice. Then we focused on mouse endogenous murine Aβ‐related peptides in the evaluation of plasma biomarker for the amyloid deposition in the brains of APP/PS1 mice. The analysis of APP/PS1 mouse plasma showed that murine APP669‐711/Aβ1‐42 and APP669‐711/Aβ1‐40 ratios were increased in old mice with plaques compared to young mice without them. However, the murine Aβ1‐40/Aβ1‐42 ratio was not changed between old and young mice. Conclusion: These results suggested that the APP669‐711/Aβ1‐42 ratio is a common biomarker for the amyloid plaque pathology in humans and APP/PS1 mice. Also, we revealed the possibility that the APP669‐711/Aβ1‐40 ratio is a biomarker specific for the brain Aβ deposition in APP/PS1 mice. These biomarkers can be useful tools to study lifestyle intervention and drug development for the prevention of amyloid deposition using the model mouse. [ABSTRACT FROM AUTHOR]

Background: Amyloid‐β peptide (Aβ) is deposited in the brains of Alzheimer disease (AD) patients, and proteolytically derived from its precursor protein, APP. APP669‐711 (a.k.a. Aβ(‐3)‐40) is a novel APP‐derived peptide detected in the plasma using immunoprecipitation‐matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (IP‐MS). We have reported that the composite plasma biomarker, which is combination of APP669‐711/Aβ1‐42 ratio and Aβ1‐40/ Aβ1‐42 ratio, surrogates the accumulation of Aβ in brain (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014; Nakamura et al., Nature 2018). However, the mechanism of APP669‐711 production is largely unclear. Method: We analyzed the effects of several inhibitors and genetic knockouts on the production of APP699‐711 in cultured cells. Result: Endogenous APP669‐711 was detected in the conditioned medium of BE2‐(C), Neuro2a and A549 cells by IP‐MS. Pharmacological experiments revealed that APP669‐711 is generated by sequential cleavages by GM6001‐sensitive metalloprotease at 669 site and γ‐secretase. Based on the preferences of the substrate sequence, we focused on ADAMTS4, which is the secreted metalloprotease with thrombospondin motif. Overexpression of ADAMTS4 resulted in the overproduction of APP669‐711, which was diminished by the catalytically inactivating mutation (i.e., E362A). Furthermore, endogenous APP669‐711 production was decreased in ADAMTS4 knockout cells. Conclusion: These results indicate that ADAMTS4 is involved in the production pathway of APP669‐711, a novel plasma biomarker for Aβ deposition in the brain. [ABSTRACT FROM AUTHOR]