Acetaminophen protects hippocampal neurons and PC12 cultures from amyloid β-peptides induced oxidative stress and reduces NF-κB activation
Introduction
The deposition and excessive accumulation of amyloid β-peptide (Aβ) is the major pathological hallmark of Alzheimer’s disease (AD), and a possible cause of neurodegeneration (Selkoe, 1996, Hardy, 1997). The main features of this pathology are the appearance of neurofibrillary tangles of hyperphosphorilated cytoskeletal protein tau and the presence of amyloid plaques, containing several peptides generated from the cleavage of the β-amyloid precursor protein (APP) by β- and γ-secretases (Coulson et al., 2000). The mechanism by which Aβ induces neuronal death is still unclear. Disruption of calcium homeostasis (Hedin et al., 2001, Mattson et al., 1992, Mattson et al., 1993, Scorziello et al., 1996) and membrane potential (Mark et al., 1995), as well as upregulation of actin polymerization (Furukawa and Mattson, 1995) have been observed following Aβ toxicity. After all, Aβ, through interaction with not yet identified targets on the cell surface (Busciglio et al., 1992, Pike et al., 1992), initiates a cascade of intracellular events that culminates in neuronal death (Forloni et al., 1993, Loo et al., 1993, Scorziello et al., 1996).
Apoptosis has been associated with the pathophysiology of AD (Smale et al., 1995, Su et al., 1994), and evidence of Aβ-induced expression of several immediate early genes, such as c-jun and c-fos, has been reported (Estus et al., 1997). Moreover, many of the genes newly induced in AD are under immediate-early transcriptional control of NF-κB, both in neurons and in microglia (Kaltschmidt et al., 1994a, Kaltschmidt et al., 1994b). This leads to the expression of the main components of the inflammatory reaction, such as the acute phase and the cellular-mediated response, and also of the cellular antioxidant system, but is also linked to the pathological evolution of amyloid deposition, being thus a crucial regulator of the cellular fate (Kaltschmidt et al., 1997).
Recently, oxidative stress showed to play a primary role in the mechanistic evaluation of Aβ-induced neuronal death (Markesbery, 1997, Markesbery and Carney, 1999, Butterfield et al., 1999, Smith et al., 1998), being the brains of AD patients under increased oxidative injury. Moreover, NF-κB is recognized as a redox-sensitive transcription factor, in that it is implicated in the cellular response to oxidative stress (Mercurio and Manning, 1999). Again, a direct relationship between oxidative stress and mitochondrial abnormalities in AD has been demonstrated (Hirai et al., 2001). In fact, AD brain areas with increased oxidative damage showed also a significant increase in mitochondrial DNA and cytochrome oxidase associated with reduced number of mitochondria. Different antioxidants, from propyl gallate (PG) to Vitamin E, have been analyzed but, at present, they do not provide substantial protection against Aβ peptides, being just able to partly attenuate neuronal damage (Pike et al., 1997, Behl et al., 1992, Behl et al., 1994).
As already mentioned, inflammation and glial activation by amyloid-derived peptides play a fundamental role in the complex array of events that leads to neurodegeneration in AD (Canning et al., 1993, Martin and O’Callaghan, 1996, Pike et al., 1995). Activated microglia and astrocytes, the increased expression of various cytokines, complement activation products and apolipoprotein E (apoE) isoforms are typically present around amyloid plaques and dystrophic neurites (Cedazo-Minguez et al., 2001, Emmerling et al., 2000), thus suggesting the development of therapeutic strategies based on anti-inflammatory drugs. Many epidemiological studies have shown the capability of indomethacin or ibuprofen to reduce the risk of AD (McGeer et al., 1996, McGeer and McGeer, 1999). Moreover, non-steroidal anti-inflammatory drugs (NSAIDs), unselective cyclooxygenase (COX) inhibitors, and selective COX-2 inhibitors are under investigation, as they could potentially reduce neuronal prostaglandin production, besides acting on astroglial and microglial cells (McGeer, 2000, Flynn and Theesen, 1999, Hull et al., 2000). Among the latest molecules tested, acetaminophen (paracetamol) has shown the capability to modulate the release of inflammatory molecules like PGE2 and IL6 by Aβ-stimulated astrocytes (Landolfi et al., 1998). Albeit some authors hypothesize the ability of acetaminophen to stimulate free radical activity as a causative agent of tardive AD (Jones, 2001), nevertheless the antioxidant properties of this atypical anti-inflammatory drug are directly linked to the capability of inhibiting lipid peroxidation (Porubek et al., 1987). This indicates that the drug is able to interfere with, at least, two different cellular pathways that result activated during inflammation, the production of inflammatory cytokines and the generation of reactive oxygen species (ROS).
From these premises, we sought to investigate if acetaminophen can reduce the apoptotic degeneration induced by amyloid derivatives on primary hippocampal neurons and on PC12, a sympathetic-derived cell line, and to analyze the role of the antioxidant properties of the drug in Aβ-triggered neuronal apoptosis. Moreover, we studied the capability of the drug to interfere with Aβ-induced activation of NF-κB transcription factor, in order to analyze the role of this drug in neuronal transcriptional activity under Aβ toxicity.
Section snippets
Materials
[Pyr3]-Amyloid β-protein (human, 3-42) was from Peptides International (Louisville, KY), amyloid β-protein (1-42) was from Quality Controlled Biochemicals (Hopkinton, MA), amyloid β-protein (1-40) was from Biosource International (Camarillo, CA). Acetaminophen was kindly provided by ACRAF S.p. A (Pomezia, Italy). Mouse nerve growth factor (m-NGF) was from Alomone Labs (Tel Aviv, Israel). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ferrous sulfate (FeSO4·7H2O), propyl
Acetaminophen rescues neuronal cells from mitochondrial redox impairment
A 24 h pre-treatment with 100 μM acetaminophen completely rescues differentiated PC12 from mitochondrial impairment by 1 μM Aβ(1-42) and Aβ(1-40), as assessed by MTT test, and acetaminophen per se does not affect PC12 survival (Fig. 1A). Preliminary experiments on peptides toxicity on hippocampal cultures were performed to find the concentrations of the peptides which impaired cell survival to a similar extent (data not shown). Aβ(1-40) was able to induce a marked neuronal toxicity at 25 μM (∼60%,
Discussion
The present data show that different amyloid β-peptides exert a progressive toxicity on both the neuronally-differentiated pheocromocytoma cell line PC12 and on primary hippocampal neurons. This toxicity leads to apoptotic death in both models and is accompanied by overproduction of reactive oxygen intermediates (ROIs). Moreover, in hippocampal neurons, Aβ(1-40) readily activates the transcription factor NF-κB. The application of the atypical non-steroidal anti-inflammatory drug acetaminophen
Acknowledgements
The financial support of ACRAF, MURST PRIN 2000, MISAN Finalizzato 99 “Invecchiamento cerebrale…” and Telethon Grant E1144 to GS is greatly appreciated.
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