MAB1563 Sigma-AldrichAnti-Presenilin-1 Antibody, NT, clone hPS1-NT

The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.

The structure and function of the gamma-secretase proteases are of great interest because of their crucial roles in cellular and disease processes. We established a novel purification protocol for the gamma-secretase complex that involves a conformation- and complex-specific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the gamma-secretase structure and its conformational variability. Under steady-state conditions, the complex adopts three major conformations, which differ in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate-binding sites by inhibitors differentially stabilizes subpopulations of particles with compact conformations, whereas a mutation linked to familial Alzheimer disease results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the csecretase complex as a dynamic population of interconverting conformations, involving rearrangements at the nanometer scale and a high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect amyloid beta (Abeta) generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely to be instrumental for highly allosteric behavior of the enzyme.

Composed of Ginsenoside Rg1 and Geniposide, the herbal medicine TongLuoJiuNao (TLJN) injection liquid has anti-inflammatory properties and can improve learning and memory in mice. Recently, TLJN has been used to treat the patients with cerebral ischemic stroke and vascular dementia, which significantly increase the risk of developing Alzheimer's disease (AD) in the early human beings. Although beneficial effects of TLJN have been reported in the vascular-associated brain disorders, the roles of TLJN in AD brains are still not clear. In this study, we chronically administered TLJN in amyloid precursor protein (APP) Swedish mutant transgenic mice (APP23) from 6 months old of age, which is at the onset of Aβ plaques, to 12 months old. We found that TLJN significantly decreased Aβ production and deposition in the brain of APP23 mice. Furthermore, we observed that TLJN down-regulated the levels and activity of β-secretase 1 (BACE1) protein as well as the expression levels of γ-secretase complex components: PS1, nicastrin and anterior pharynx-defective 1 (APH1) but not presenilin enhancer 2 (PEN2). The results suggest an inhibitory effect of TLJN on amyloidogenic APP processing by down-regulating the cleavage enzymes BACE1 and γ-secretase.

Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q.We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro.Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease.

Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aβ) generation as well. Here we demonstrate that the secretion of Aβ is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO(-)), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aβ towards Aβ(42) and increased the Aβ(42) /Aβ(40) ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair γ-secretase and 'loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ɛ-cleavage function is not generally observed among FAD mutants. On the other hand, γ-secretase inhibitors used in the clinic appear to block the initial ɛ-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer's disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Huperzine A (HupA) is a reversible and selective inhibitor of acetylcholinesterase (AChE), and it has multiple targets when used for Alzheimer's disease (AD) therapy. In this study, we searched for new mechanisms by which HupA could activate Wnt signaling and reduce amyloidosis in AD brain. A nasal gel containing HupA was prepared. No obvious toxicity of intranasal administration of HupA was found in mice. HupA was administered intranasally to β-amyloid (Aβ) precursor protein and presenilin-1 double-transgenic mice for 4 months. We observed an increase in ADAM10 and a decrease in BACE1 and APP695 protein levels and, subsequently, a reduction in Aβ levels and Aβ burden were present in HupA-treated mouse brain, suggesting that HupA enhances the nonamyloidogenic APP cleavage pathway. Importantly, our results further showed that HupA inhibited GSK3α/β activity, and enhanced the β-catenin level in the transgenic mouse brain and in SH-SY5Y cells overexpressing Swedish mutation APP, suggesting that the neuroprotective effect of HupA is not related simply to its AChE inhibition and antioxidation, but also involves other mechanisms, including targeting of the Wnt/β-catenin signaling pathway in AD brain.

gamma-Secretase is an intramembrane-cleaving protease responsible for the final proteolytic event in the production of the amyloid-beta peptides (Abeta) implicated in Alzheimer's disease (AD). Inhibition of gamma-secretase activity is thus an attractive therapeutic strategy to slow down the pathogenesis of AD. Drugs often target more than one biomolecule because of conserved 3-dimensional structures in prospective protein binding sites. We have capitalized on this phenomenon of nature to identify new gamma-secretase inhibitors. Here we show that 2-hydroxy naphthyl derivatives, a previously identified subclass of NAD(+) analog inhibitors of sirtuin 2 (SIRT2), are direct gamma-secretase inhibitors. Subsequent structure-activity relationship studies further showed that 2-hydroxy-1-naphthaldehyde is the minimal pharmacophore for gamma-secretase inhibition. In evaluating target protein determinants of inhibition, we identified a common GXG signature nucleotide-binding site (NBS) shared by the gamma-secretase subunit presenilin-1 C-terminal fragment (PS1-CTF), SIRT2, and Janus kinase 3 (JAK3). Because a detailed 3-dimensional structure of gamma-secretase is beyond our knowledge, we took advantage of the known crystal structure of human JAK3 to model the NBS of the PS1-CTF, which includes the catalytic residue D385. Our results suggest that the flexible PS1-CTF (381)LGLG(384) loop comprises a substrate-docking site capable of recognizing specifically different gamma-secretase substrates.-Wu, F., Schweizer, C., Rudinskiy, N., Taylor, D. M., Kazantsev, A., Luthi-Carter, R., Fraering, P. C. Novel gamma-secretase inhibitors uncover a common nucleotide-binding site in JAK3, SIRT2, and PS1.

Abnormal zinc homeostasis is involved in β-amyloid (Aβ) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer's disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aβ deposition has not been well established in AD animal models in vivo.In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro.The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.