Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer's disease.
Por:
Boix CP, Lopez-Font I, Cuchillo-Ibañez I and Sáez-Valero J
Publicada:
12 ago 2020
Ahead of Print:
12 ago 2020
Resumen:
BACKGROUND: The amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes alternative proteolytic processing. Its processing through the amyloidogenic pathway originates a large sAPPß ectodomain fragment and the ß-amyloid peptide, while non-amyloidogenic processing generates sAPPa and shorter non-fibrillar fragments. Hence, measuring sAPPa and sAPPß 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 sAPPa and sAPPß fragments and their membrane-tethered C-terminal fragments CTFa and CTFß. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether Aß42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPPa and sAPPß 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 sAPPa or sAPPß protein levels remained unchanged, as did those of CTFa and CTFß. We studied the glycosylation of the brain sAPPa and sAPPß 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 sAPPß species derived from the APP695 and APP-KPI variants, probably reflecting their distinct cellular origin. Moreover, the lectin-binding pattern differed in the sAPPa and sAPPß originated from all the variants. Finally, when the lectin-binding pattern was compared between AD and NDC groups, significant differences were evident in sAPPa glycosylation. Lectin binding of the soluble sAPPa and sAPPß from CHO-PS70 cells were also altered in cells treated with the Aß peptide. CONCLUSION: Our analysis of the lectin binding to sAPPa and sAPPß 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.
Filiaciones:
:
Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Av. Ramón y Cajal s/n, E-03550, Sant Joan d'Alacant, Spain
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Sant Joan d'Alacant, Spain
:
Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Av. Ramón y Cajal s/n, E-03550, Sant Joan d'Alacant, Spain.
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Sant Joan d'Alacant, Spain.
Instituto de Investigacion Sanitaria y Biomedica de Alicante (ISABIAL), Alicante, Spain.
:
Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Av. Ramón y Cajal s/n, E-03550, Sant Joan d'Alacant, Spain
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Sant Joan d'Alacant, Spain
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