Abstract
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of β-amyloid (Aβ) peptides. Aβ is a cleavage product of the amyloid precursor protein (APP), and aberrant posttranslational modifications of APP can alter APP processing and increase Aβ generation. In the AD brain, seven different residues, including Ser-675 (APP695 numbering) in the APP cytoplasmic domain has been found to be phosphorylated. Here, we show that expression of a phosphomimetic variant of Ser-675 in APP (APP-S675E), in human neuroblastoma SK-N-AS cells, reduces secretion of the soluble APP ectodomain (sAPPα), even though the total plasma membrane level of APP was unchanged compared with APP levels in cells expressing APPwt or APP-S675A. Moreover, the level of an alternative larger C-terminal fragment (CTF) increased in the APP-S675E cells, whereas the CTF form that was most abundant in cells expressing APPwt or APP-S675A decreased in the APP-S675E cells. Upon siRNA-mediated knockdown of the astacin metalloprotease meprin β, the levels of the alternative CTF decreased and the CTF ratio was restored back to APPwt levels. Our findings suggest that APP–Ser-675 phosphorylation alters the balance of APP processing, increasing meprin β–mediated and decreasing α-secretase–mediated processing of APP at the plasma membrane. As meprin β cleavage of APP has been shown to result in formation of highly aggregation-prone, truncated Aβ2–40/42 peptides, enhanced APP processing by this enzyme could contribute to AD pathology. We propose that it would be of interest to clarify in future studies how APP–Ser-675 phosphorylation promotes meprin β–mediated APP cleavage.
Highlights
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of -amyloid (A) peptides
A prominent sAPP␣ secretion was detected from APPwt and amyloid precursor protein (APP)-S675A cells, a reduction of sAPP␣ secretion by ϳ50% was observed from APP-S675E cells (Fig. 1, A and D)
In this study we for the first time show that phosphorylation of APP–Ser-675, a phosphorylation known to occur in AD brain [15], can regulate the processing of APP
Summary
APP–Ser-675 phosphorylation decreases sAPP␣ secretion while increasing the level of a slower migrating APP-CTF. Analysis of total sAPP secretion from APPwt, APPS675A, and APP-S675E cells, using the 22C11 antibody, showed that the secretion of total sAPP from APP-S675E cells was reduced to the same extent as the sAPP␣ secretion (Fig. 1, A and E) Taken together this suggests that the slower migrating CTF in APP-S675E cells is likely C99* generated by meprin . The total level of CTFs did not change (Fig. 5D); instead, meprin  knockdown returned the ratio between the upper and lower CTF in the APP-S675E cells back toward the ratio observed in APPwt cells (Fig. 5F) These data suggest that meprin  is responsible for the generation of the slower migrating CTF and that phosphorylation of Ser-675 in APP695 shifts the balance between ␣-secretase and meprin  processing of APP, favoring meprin  cleavage. This suggests that ADAM10 could be involved in the generation of the slower migrating CTF
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