Impairments in Aβ removal are increasingly being considered as a possible cause for the abnormal Aβ build-up typical of Alzheimer's disease (AD). Of particular interest is a pool of Aβ that accumulates intraneuronally and may contribute to neuronal toxicity. The mechanism for intraneuronal accumulation, however, is not well understood and is commonly attributed to impaired removal of extracellular Aβ by neurons. Based on the intracellular distribution of the well-established Aβ degrading enzymes, ECE-1 and ECE-2, we tested whether impairments in their catalytic activity could lead to intracellular Aβ accumulation. Using SH-SY5Y cells overexpressing wild-type APP and pharmacological inhibition of endogenous ECE activity, we found that ECEs participate in the degradation of at least 2 distinct pools of Aβ; one destined for secretion and the other being produced and degraded within the endosomal-autophagic-lysosomal pathways. While ECE-1 regulates both pools of Aβ, ECE-2 regulates mainly the intracellular pool of the peptide. Consistent with this result, ECE-2 was found to co-localize with markers of the endosomal/lysosomal pathway, but not with a TGN marker. Furthermore, ECE-2 was detected in autophagic vesicles in cells treated with chloroquine. Under these conditions, ECE inhibition produced significantly higher elevations in intracellular Aβ than chloroquine treatment alone. This study highlights the existence of Aβ clearance mechanisms by ECEs at intracellular sites of production. Alterations in ECE activity may be considered as a cause for increased intraneuronal Aβ in AD.
"It is now becoming more evident that Aβ levels in the brain are manipulable due to a dynamic equilibrium between its production from APP and removal by a cohort of amyloid clearance proteins which can be either of enzymic nature (proteases ) or non-enzymic (binding/transport proteins) (Figure 1). The number of enzymes capable of proteolytic degradation of Aβ discovered to date is rapidly growing and currently the family of amyloid-degrading enzymes (ADEs) embrace more than 20 members both membrane-bound and soluble, and of differing cellular and subcellular locations (for review see Marr and Spencer, 2010; Nalivaeva et al., 2012a,c; Grimm et al., 2013; Pacheco-Quinto et al., 2013). Intriguingly several of the main ADEs are members of the M13 peptidase family (neprilysin (NEP), NEP2 and endothelin converting enzymes (ECE-1 and ECE-2)). "
[Show abstract][Hide abstract] ABSTRACT: Abnormal elevation of amyloid β-peptide (Aβ) levels in the brain is the primary trigger for neuronal cell death specific to Alzheimer's disease (AD). It is now evident that Aβ levels in the brain are manipulable due to a dynamic equilibrium between its production from the amyloid precursor protein (APP) and removal by amyloid clearance proteins. Clearance can be either enzymic or non-enzymic (binding/transport proteins). Intriguingly several of the main amyloid-degrading enzymes (ADEs) are members of the M13 peptidase family (neprilysin (NEP), NEP2 and the endothelin converting enzymes (ECE-1 and -2)). A distinct metallopeptidase, insulin-degrading enzyme (IDE), also contributes to Aβ degradation in the brain. The ADE family currently embraces more than 20 members, both membrane-bound and soluble, and of differing cellular locations. NEP plays an important role in brain function terminating neuropeptide signals. Its decrease in specific brain areas with age or after hypoxia, ischaemia or stroke contribute significantly to the development of AD pathology. The recently discovered mechanism of epigenetic regulation of NEP (and other genes) by the APP intracellular domain (AICD) and its dependence on the cell type and APP isoform expression suggest possibilities for selective manipulation of NEP gene expression in neuronal cells. We have also observed that another amyloid-clearing protein, namely transthyretin (TTR), is also regulated in the neuronal cell by a mechanism similar to NEP. Dependence of amyloid clearance proteins on histone deacetylases and the ability of HDAC inhibitors to up-regulate their expression in the brain opens new avenues for developing preventive strategies in AD.
"There is evidence suggesting that the soluble oligomers have a higher neurotoxicity than monomers and are responsible for synaptic degradation [7e9]. Several enzymes that are known to hydrolyze amyloid beta have also been shown to regulate the endogenous amyloid beta concentration, such as neprilysin (NEP)  , endothelin converting enzyme (ECE)-1   and insulin degrading enzyme (IDE)  . The Ab degradation has been determined by sandwich enzymelinked immunosorbent assay (ELISA) or western blot methods, in which the decline in Ab 1-42 and/or Ab 1-40 concentration over time is measured  . "
[Show abstract][Hide abstract] ABSTRACT: Deposition of insoluble amyloid plaques is one of the known hallmarks of Alzheimer's disease. Amyloid beta 1-42 is the main component in these plaques, and the soluble oligomers of this peptide are believed to contribute to synaptic degradation and dementia. Enzymatic hydrolysis of amyloid beta is important to keep its tissue concentration low to avoid oligomerization. We have employed four enzymes involved in in vivo degradation of amyloid beta, to identify amyloid beta 1-42 hydrolysis products in vitro. Liquid chromatography coupled to (high resolution) mass spectrometry was used to identify the proteolysis products. Novel cleavage sites were discovered for all four enzymes. For each enzyme, the peptide was incubated for several different periods from 0.5 to 210 min, and the proteolysis products from each period were characterized. Thus, both the initial cleavage sites and the full degradation profiles were revealed. Knowledge about the fate of amyloid beta is important to better understand the mechanism underlying Alzheimer`s disease, and the reported proteolysis products can be used as targets in future investigations on amyloid beta clearance.
"SH-SY5Y-APP cells were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F12 (DMEM-F12) (Thermo scientific) medium with 10% heat inactivated FBS and 1% penicillin-streptomycin as described previously . Cells were plated at 3×105 cells per well in 6 well dishes and incubated for 24 h in DMEM-F12-10% FBS medium. "
[Show abstract][Hide abstract] ABSTRACT: BACE1 (β-secretase) and α-secretase cleave the Alzheimer's amyloid β protein (Aβ) precursor (APP) to C-terminal fragments of 99 aa (CTFβ) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aβ and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing Aβ to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aβ in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aβ40 and Aβ42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aβ, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFβ, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aβ degrading activity, endothelin converting enzyme (ECE), yielded more Aβ, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aβ by transiently skirting Aβ degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.
PLoS ONE 03/2014; 9(3):e91531. DOI:10.1371/journal.pone.0091531 · 3.23 Impact Factor
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