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 nonfibrillar 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.

Amyloid aggregation of Streptococcus mutans Cnm influences its collagen-binding activity

“…The oligomerization of Cnm into amyloids does not seem to be dependent upon glycosylation by the Pgf machinery, since native glycosylated and non-glycosylated recombinant proteins both activated ThT fluorescence and displayed visible amyloid fibers when visualized by microscopy. The contribution of glycosylation to amyloidogenic proteins is still a relatively unexplored field, although the effect of glycosylation of the Amyloid Precursor Protein of Aβ in Alzheimer's disease has gained attention due to its regulatory role in the protein's proteolytic processing (56,57). Bacterial amyloids derived from glycoproteins have now been reported as components of the structural extracellular matrix of biofilms rich in ammonia-oxidizing bacteria and nitrite-oxidizing bacteria grown in aerobic and granular sludge (58).…”

Exploring the Efficacy of Anti-amyloid-β Therapeutics in Treating Alzheimer Disease

“…Secretase enzymes function in the alternative proteolytic processing of APP (reviewed in [33]). It is the proteolytic processing of the ubiquitous glycoprotein APP that generates the Aβ peptide [34]. The metabolism of APP is usually carried out by an extracellular protease α-secretase and an intramembrane protease γ-secretase, creating a soluble protein that can be broken down to aid in neuron repair [2].…”

“…The metabolism of APP is usually carried out by an extracellular protease α-secretase and an intramembrane protease γ-secretase, creating a soluble protein that can be broken down to aid in neuron repair [2]. This is so-called the non-amyloidogenic pathway, and it co-exists with the pathologic (abnormal) amyloidogenic pathway in which the toxic Aβ peptide is formed [34]. The sequential cleavage of the APP with γ-secretase and β-secretase, also known as β-site APP cleaving enzyme 1 (BACE1), gives rise to Aβ [1,2].…”

Mitochondria-associated membranes (MAMs): a potential therapeutic target for treating Alzheimer’s disease

“…Aβ is a self-aggregating peptide containing 40–42 amino acids that is generated by the proteolytic processing of APP, a ubiquitous glycoprotein generally expressed in the whole brain. Full-length APP (700 amino acids) is composed of a large transmembrane N-terminal ectodomain including the Aβ sequence and a short intracellular C-terminal domain [ 14 ]. APP proteolytic processing occurs via three pathways ( Figure 1 ): (1) The non-amyloidogenic pathway.…”