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Alzheimer's disease (AD) is caused by the accumulation of neurotoxic amyloid-β (Aβ) peptides. Aβ is derived from amyloid-β protein precursor (AβPP). In the non-amyloidogenic pathway, AβPP is cleaved by α-secretase and γ-secretase at the plasma membrane, excluding Aβ production. Alternatively, AβPP in the plasma membrane is internalized via endocytosis, and delivered to early endosomes and lysosomes, where it is cleaved by β-secretase and γ-secretase. Recent studies have shown that insulin in the periphery crosses the blood-brain barrier, and plays important roles in the brain. Furthermore, impaired insulin signaling has been linked to the progression of AD, and intranasal insulin administration improves memory impairments and cognition. However, the underlying molecular mechanisms of insulin treatment remain largely unknown. To investigate the effects of insulin on AβPP processing, we tested the effects of insulin on neuroblastoma SH-SY5Y cells overexpressing AβPP, and cultured rat cortical neurons. We found that insulin increased the level of cell surface AβPP, decreasing the endocytosis rate of AβPP. Insulin reduced Aβ generation through upregulation of AβPP O-GlcNAcylation via Akt insulin signaling. Our present data suggest that insulin affects Aβ production by regulating AβPP processing through AβPP O-GlcNAcylation. These results provide mechanistic insight into the beneficial effects of insulin, and a possible link between insulin deficient diabetes and cerebral amyloidosis in the pathogenesis of AD. [ABSTRACT FROM AUTHOR]

Familial Alzheimer’s disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of β-amyloid (Aβ) from β-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aβ production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-β-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts. [ABSTRACT FROM AUTHOR]

The pathological hallmark of Alzheimer's disease (AD) is associated with the accumulation of amyloid-β (Aβ) derived from proteolytic processing of amyloid-β precursor protein (APP). APP undergoes post-translational modification including N- and O-glycosylation. O-GlcNAcylation is a novel type of O-glycosylation, mediated by O-GlcNAc transferase attaching O-β- N -acetylglucosamine (O-GlcNAc) to serine/threonine residues of the target proteins. O-GlcNAc is removed by O-GlcNAcase. We have previously reported that increasing O-GlcNAcylated APP using the O-GlcNAcase inhibitor, PUGNAc, increases its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased Aβ production. However, O-GlcNAc modification sites in APP are unknown. In this study, we mutated three predicted O-GlcNAc modification threonine residues of APP into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. These APP mutants showed reduced O-GlcNAcylation levels, indicating that these sites were endogenously O-GlcNAcylated. Thr 576 was the major O-GlcNAcylation site when cell was treated with PUGNAc. We also showed that the effects of PUGNAc on APP trafficking to the plasma membrane and Aβ production were prevented in the T576A mutant. These results implicate Thr 576 as the major O-GlcNAcylation site in APP and indicate that O-GlcNAcylation of this residue regulates its trafficking and processing. Thus, specific O-GlcNAcylation of APP at Thr 576 may be a novel and promising drug target for AD therapeutics. [ABSTRACT FROM AUTHOR]

Like protein phosphorylation, O-GlcNAcylation is a common post-translational protein modification. We already reported that O-GlcNAcylation of amyloid precursor protein (APP) in response to insulin signaling reduces neurotoxic amyloid-β (Aβ) production via inhibition of APP endocytosis. Internalized APP is delivered to endosomes and lysosomes where Aβ is produced. However, the molecular mechanism involved in the effect of APP O-GlcNAcylation on APP trafficking remains unknown. To investigate the relationship between APP O-GlcNAcylation and APP endocytosis, we tested the effects of insulin on neuroblastoma SH-SY5Y cells overexpressing APP and BACE1, and cultured rat hippocampal neurons. The present study showed that APP O-GlcNAcylation translocated APP from lipid raft to non-raft microdomains in the plasma membrane by using immunocytochemistry and discontinuous sucrose gradients method. By using the biotinylation method, we also found that APP preferentially underwent endocytosis from lipid rafts and that the amount of internalized APP from lipid rafts was specifically reduced by O-GlcNAcylation. These results indicate that O-GlcNAcylation can regulate lipid raft-dependent APP endocytosis via translocation of APP into non-raft microdomains. Our findings showed a new functional role of O-GlcNAcylation for the regulation of APP trafficking, offering new mechanistic insight for Aβ production. [ABSTRACT FROM AUTHOR]

Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-β (Aβ) is produced via β-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aβ generation. β-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer's disease. [ABSTRACT FROM AUTHOR]