Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer's disease

Abstract

Background: The amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes alternative proteolytic processing. Its processing through the amyloidogenic pathway originates a large sAPPβ ectodomain fragment and the β-amyloid peptide, while non-amyloidogenic processing generates sAPPα and shorter non-fibrillar fragments. Hence, measuring sAPPα and sAPPβ has been proposed as a means to identify imbalances between the amyloidogenic/non-amyloidogenic pathways in the brain of Alzheimer's disease (AD) patients. However, to date, no consistent changes in these proteolytic fragments have been identified in either the brain or cerebrospinal fluid of AD individuals.

Methods: In frontal cortex homogenates from AD patients (n = 7) and non-demented controls (NDC; n = 7), the expression of total APP mRNA and that of the APP isoforms generated by alternative splicing, APP695 and APP containing the Kunitz protease inhibitor (KPI), was analyzed by qRT-PCR using TaqMan and SYBR Green probes. The balance between the amyloidogenic/non-amyloidogenic pathways was examined in western blots estimating the sAPPα and sAPPβ fragments and their membrane-tethered C-terminal fragments CTFα and CTFβ. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether Aβ42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPPα and sAPPβ in brain extracts and CHO-PS70 culture media by lectin-binding assays.

Results: In the cortex of AD patients, we detected an increase in total APP mRNA relative to the controls, due to an increase in both the APP695 and APP-KPI variants. However, the sAPPα or sAPPβ protein levels remained unchanged, as did those of CTFα and CTFβ. We studied the glycosylation of the brain sAPPα and sAPPβ using lectins and pan-specific antibodies to discriminate between the fragments originated from neuronal APP695 and glial/KPI variants. Lectin binding identified differences in the glycosylation of sAPPβ species derived from the APP695 and APP-KPI variants, probably reflecting their distinct cellular origin. Moreover, the lectin-binding pattern differed in the sAPPα and sAPPβ originated from all the variants. Finally, when the lectin-binding pattern was compared between AD and NDC groups, significant differences were evident in sAPPα glycosylation. Lectin binding of the soluble sAPPα and sAPPβ from CHO-PS70 cells were also altered in cells treated with the Aβ peptide.

Conclusion: Our analysis of the lectin binding to sAPPα and sAPPβ suggests that glycosylation dictates the proteolytic pathway for APP processing. Differences between the demented and controls indicate that changes in glycosylation may influence the generation of the different APP fragments and, consequently, the pathological progression of AD.