N-Acetylcysteine Prevents 4-Hydroxynonenal- and Amyloid-beta-Induced Modification and Inactivation of Neprilysin in SH-SY5Y Cells
ABSTRACT As one of the dominant amyloid-beta peptide (Abeta) proteases, neprilysin (NEP) plays a crucial role in maintaining a physiologic balance between Abeta production and catabolism. We have previously shown that NEP is modified by 4-hydroxynonenal (HNE) adducts, resulting in decreased activity in the brain of AD patients and cultured cells. In order to determine whether antioxidants can rescue NEP, SH-SY5Y cells were treated with HNE or Abeta, together with N-acetylcysteine for 24 hours, prior to analysis of NEP protein levels, activity, and oxidative modifications. Intracellular NEP developed HNE adducts after 24 hours of HNE or Abeta treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. N-acetylcysteine at 10 to 100 microM alleviated HNE adduction after HNE or Abeta treatment. In keeping with previous reports, HNE-modified NEP showed decreased catalytic activity. The present study demonstrates that antioxidants can be used to spare NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in aging or Alzheimer's disease.
SourceAvailable from: Giuseppe Grasso[Show abstract] [Hide abstract]
ABSTRACT: In recent years there has been a wide interest toward zinc metalloproteases involved in neurodegenerative diseases. Enzymes such as matrix metalloproteases, neprilysin, insulin degrading enzyme, etc. play a pivotal role in the homeostasis of peptides and proteins whose accumulation and fibrillation seem to be the major cause for the development of such diseases. It is also well known that chemical factors such as oxidative stress, small molecules and metal ions are able to significantly affect the activity of the above mentioned metalloproteases and therefore, very recently, many research groups have been focused on studying the interaction between those chemical factors and some of the enzymes involved in neurodegenerative diseases. Particularly, metal ions can play various roles (catalytic, structural, allosteric) in enzymes and both the coordination environment as well as the specific metal ion involved, determine the particular function of a metalloprotease. In this article, chemical factors which modulate the activity of zinc metalloproteases involved in neurodegenerative diseases are examined together with recently proposed therapeutic routes related to them. Moreover, two of the most used experimental techniques applied for studying such modulation are also reviewed.Coordination Chemistry Reviews 02/2014; 260:139–155. DOI:10.1016/j.ccr.2013.10.008 · 12.10 Impact Factor
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ABSTRACT: Senile plaques composed of β-amyloid (Aβ) are a pathological hallmark of Alzheimer disease. Presenilin 2 (PS2) mutations increase Aβ generation in the brains of Alzheimer disease patients, but the underlying mechanism of Aβ generation by PS2 mutations remains to be clarified. The Aβ is generated through the sequential cleavage of amyloid precursor protein by β- and γ-secretases. Here, we show that the PS2 mutation N141I enhances the activity of β-secretase and expression of the β-site amyloid precursor protein cleavage enzyme 1, a major neuronal β-secretase in the brains of PS2 transgenic mice and in PC12 cells overexpressing mutant PS2. In parallel with the increased activity of β-secretase, activation of extracellular signal-regulated kinase (ERK), Aβ1-40 and Aβ1-42 levels, generation of reactive oxygen species, and lipid peroxidation were higher in the mutant mouse neurons and the PC12 cells. Colocalization of phosphorylated ERK (phospho-ERK) and β-site amyloid precursor protein cleavage enzyme 1 with hydroxynonenal-histidine was found in the mutant brains. An ERK inhibitor U0126 and an antioxidant N-acetylcysteine prevented the expression and activity of β-secretase, ERK activation, and reactive oxygen species generation in both neurons and PC12 cells expressing mutant PS2 in a dose-dependent manner. Together, these data suggest that oxidative stress-mediated ERK activation contributes to increases in β-secretase and, thus, an increase of Aβ generation in neuronal cells expressing mutant PS2.Journal of Neuropathology and Experimental Neurology 02/2012; 71(2):130-9. DOI:10.1097/NEN.0b013e3182432967 · 4.37 Impact Factor
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ABSTRACT: We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD.American Journal of Translational Research 01/2013; 5(6):608-621. · 3.23 Impact Factor