No abstract

␥-Secretase is an enzyme complex that mediates both Notch signaling and ␤-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid ␤ peptide (A␤), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of ␥-secretase gives rise to A␤ peptides of different lengths, where A␤42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320 -374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of ␥-secretase, for ␥-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, ␥-secretase complex formation, and had a differential effect on A␤-peptide production. Although the production of A␤38, A␤39, and A␤40 was severely impaired, the effect on A␤42 was affected to a lesser extent, implying that the production of the AD-related A␤42 peptide is separate from the production of the A␤38, A␤39, and A␤40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ⑀/S3 and ␥ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different A␤ peptides without affecting Notch processing, two parameters of significance when considering ␥-secretase as a target for pharmaceutical intervention in AD.

SummaryWhen a chromogenicLimulusAmoebocyte Lysate (LAL) assay and a tube gelation LAL assay were compared for the detection of Gram-negative bacteria, using a strain ofPseudomonas putida, the detection level (∼ 103cfu/ml) and cost of the assays were approximately the same for both assays but the reading was more precise for the chromogenic substrate assay. A modified chromogenic assay was devised for detection ofPs. putidain milk.

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer’s disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ40 and Aβ42 production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ42 accompanied by increased intracellular Aβ42/Aβ40 ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ42/Aβ40 ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ42/Aβ40 ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.

Background: In the intensive ongoing study to elucidate the relationship between the hallmarks of Alzheimer's disease (AD), the reciprocal interaction between amyloid metabolism and the cholinergic enzyme acetylcholinesterase (AChE) has been addressed and various levels of interactions have been suggested. We have previously identified presenilin-1 (PS1), the active component of the g-secretase complex, as an interacting protein of AChE. In this study, we have explored some of the consequences of AChE-PS1 interactions. Methods: The expression of the cholinergic AChE (also called T or "tailed") and the "read thought" (R) splicing variant was modulated in SH-SY5Y neuroblastoma cells by transgenic over-expression and by knockdown with siRNA. We also tested whether the classical agonist of acetylcholine receptors, carbachol may affect PS1 levels. Finally, we evaluated PS1 levels in amyloid Aß42-treated SH-SY5Y cells with and without AChE knock-down. Results: We showed an AChE influence on PS1 levels by showing that AChE knock-down with siRNA in SH-SY5Y neuroblastoma cells decreased PS1 levels. We found that AChE over-expression exerts opposing effects on PS1 levels, leading to increased level of PS1 protein in transfected cells. Interestingly, over-expression of the AChE-R variant, which is normally present at low levels in the mammalian brain, was more effective influencing PS1 than the over-expression of the major cholinergic AChE-T variant. The cholinergic agonist carbachol failed to exert an effect on PS1. Finally we exposed neuroblastoma cells to Aß42 which triggered elevation of both AChE and PS1 levels. The Aß42-induced PS1 increase was abolished by pre-treatment of SH-SY5Y with siRNA AChE, suggesting that AChE may participate in the pathological feed-back loop between PS1 and Aß. Conclusions: Our results provide insight into AChE-amyloid interrelationships and identify a new molecular interaction that may contribute to AD pathology and have therapeutic implications.