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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 sAPP? ectodomain fragment and the ?-amyloid peptide, while non-amyloidogenic processing generates sAPP? and shorter non-fibrillar fragments. Hence, measuring sAPP? and sAPP? 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 sAPP? and sAPP? fragments and their membrane-tethered C-terminal fragments CTF? and CTF?. CHO-PS70 cells, stably over-expressing wild-type human APP, served to evaluate whether A?42 peptide treatment results in altered APP glycosylation. We determined the glycosylation pattern of sAPP? and sAPP? 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 sAPP? or sAPP? protein levels remained unchanged, as did those of CTF? and CTF?. We studied the glycosylation of the brain sAPP? and sAPP? 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 sAPP? species derived from the APP695 and APP-KPI variants, probably reflecting their distinct cellular origin. Moreover, the lectin-binding pattern differed in the sAPP? and sAPP? originated from all the variants. Finally, when the lectin-binding pattern was compared between AD and NDC groups, significant differences were evident in sAPP? glycosylation. Lectin binding of the soluble sAPP? and sAPP? from CHO-PS70 cells were also altered in cells treated with the A? peptide. CONCLUSION:Our analysis of the lectin binding to sAPP? and sAPP? 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.

SUBMITTER: Boix CP

PROVIDER: S-EPMC7425076 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Boix Claudia P CP Lopez-Font Inmaculada I Cuchillo-Ibañez Inmaculada I Sáez-Valero Javier J

Alzheimer's research & therapy 20200812 1

<h4>Background</h4>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 ...[more]

PMID: 32787955

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Project description:INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering A? production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP ?-site cleavage (equivalent to A?40), and Notch ?-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of A? production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain A? was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain A? reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting A? production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain A? in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain A? was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.

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

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

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