Notch1 competes with the amyloid precursor protein for gamma-secretase and down-regulates presenilin-1 gene expression

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.     

The gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein induces apoptosis via Tip60 in H4 cells

The amyloid precursor protein (APP), a large glycoprotein highly expressed in neurons, is cleaved in its intramembranous domain by gamma secretase to generate amyloid-beta and a free carboxyl-terminal intracellular fragment (APP-CT), which has previously been suggested to interact with the adapter protein Fe65 and the histone acetyltransferase Tip60. An identical gamma secretase activity mediates cleavage of Notch, releasing an intracellular signaling domain that translocates to the nucleus. We examined the effect of an ectopically expressed 58-amino acid APP-CT fragment (APP-C58) on human H4 neuroglioma cells. We demonstrate by confocal microscopy and fluorescence resonance energy transfer analysis that APP-C58 translocates to the nucleus and forms a complex in the nucleus with the Tip60, independent of interactions with Fe65. APP-C58 transfected H4 cells undergo apoptosis within 48-72 h, marked by nuclear blebbing, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, and blockade by a caspase inhibitor. When nuclear access of APP-C58 is prevented by fusing with a strong membrane-targeting farnesylation domain, apoptosis is blocked. APP-C58-induced apoptosis was markedly enhanced by co-transfection with wild type Tip60 and decreased by mutant Tip60 lacking histone acetyltransferase activity, suggesting that Tip60 mediates APP-CT-induced cell death. Thus, gamma secretase cleavage of APP may contribute to Alzheimer's disease-related neurodegeneration in two ways: release of amyloid-beta and liberation of a bioactive carboxyl-terminal domain from membrane-bound APP.     

Low density lipoprotein receptor-related protein (LRP) interacts with presenilin 1 and is a competitive substrate of the amyloid precursor protein (APP) for gamma-secretase

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and the Notch receptor. Recently, the low density receptor-related protein (LRP) has been shown to be cleaved by a gamma-secretase-like activity. We postulated that LRP may interact with PS1 and tested its role as a competitive substrate for gamma-secretase. In this report we show that LRP colocalizes and interacts with endogenous PS1 using coimmunoprecipitation and fluorescence lifetime imaging microscopy. In addition, we found that gamma-secretase active site inhibitors do not disrupt the interaction between LRP and PS1, suggesting that the substrate associates with a gamma-secretase docking site located in close proximity to PS1. This is analogous to APP-gamma-secretase interactions. Finally, we show that LRP competes with APP for gamma-secretase activity. Overexpression of a truncated LRP construct consisting of the C terminus, the transmembrane domain, and a short extracellular portion leads to a reduction in the levels of the Abeta40, Abeta42, and p3 peptides without changing the total level of APP expression. In addition, transfection with the beta-chain of LRP causes an increase in uncleaved APP C-terminal fragments and a concomitant decrease in the signaling effects of the APP intracellular domain. In conclusion, LRP is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.     

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide

The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.     

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the gamma-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Abeta). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.