Acetaminophen protects hippocampal neurons and PC12 cultures from amyloid β-peptides induced oxidative stress and reduces NF-κB activation

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Abstract

The present findings show that an atypical non-steroidal anti-inflammatory drug, such as acetaminophen, retains the ability to recover amyloid β-peptides driven neuronal apoptosis through the impairment of oxidative stress. Moreover, this compound reduces the increased NF-κB binding activity, which occurs in these degenerative conditions.

Therapeutic interventions aimed at reducing the inflammatory response in Alzheimer’s disease (AD) recently suggested the application of non-steroidal anti-inflammatory drugs. Although the anti-inflammatory properties of acetaminophen are controversial, it emerged that in an amyloid-driven astrocytoma cell degeneration model acetaminophen proved to be effective.

On these bases, we analyzed the role of acetaminophen against the toxicity exerted by different Aβ-peptides on rat primary hippocampal neurons and on a rat pheochromocytoma cell line. We found a consistent protection from amyloid β-fragments 1-40 and 1-42-induced impairment of mitochondrial redox activity on both cell cultures, associated with a marked reduction of apoptotic nuclear fragmentation. An antioxidant component of the protective activity emerged from the analysis of the reduction of phospholipid peroxidation, and also from a significant reduction of cytoplasmic accumulation of peroxides in the pheochromocytoma cell line. Moreover, activation of NF-κB by amyloid-derived peptides was greatly impaired by acetaminophen pre-treatment in hippocampal cells.

This evidence points out antioxidant and anti-transcriptional properties of acetaminophen besides the known capability to interfere with inflammation within the central nervous system, and suggests that it can be exploited as a possible therapeutic approach against AD.

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.

References (73)

  • M.R. Emmerling et al.

    The role of complement in Alzheimer’s disease pathology

    Biochim. Biophys. Acta

    (2000)
  • Q. Guo et al.

    Secreted β-amyloid precursor protein counteracts the proapoptotic action of mutant presenilin-1 by activation of NF-κB and stabilization of calcium homeostasis

    J. Biol. Chem.

    (1998)
  • J. Hardy

    Amyloid, the presenilins and Alzheimer’s disease

    Trends Neurosci.

    (1997)
  • H.L. Hedin et al.

    Human platelet calcium mobilisation in response to β-amyloid (25–35): buffer dependency and unchanged response in Alzheimer’s disease

    Neurochem. Int.

    (2001)
  • G.R. Jones

    Causes of Alzheimer’s disease: paracetamol (acetaminophen) today? Amphetamines tomorrow

    Med. Hypotheses

    (2001)
  • C. Kaltschmidt et al.

    Transcription factor NF-κB is activated in microglia during experimental autoimmune encephalomyelitis

    J. Neuroimmunol.

    (1994)
  • C. Landolfi et al.

    Inflammatory molecule release by β-amyloid-treated T98G astrocytoma cells: role of prostaglandins and modulation by paracetamol

    Eur. J. Pharmacol.

    (1998)
  • W.R. Markesbery

    Oxidative stress hypothesis in Alzheimer’s disease

    Free Rad. Biol. Med.

    (1997)
  • P.J. Marshall et al.

    Constraints on prostaglandin biosynthesis in tissues

    J. Biol. Chem.

    (1987)
  • M.P. Mattson et al.

    Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium

    Brain Res.

    (1995)
  • M.P. Mattson et al.

    β-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer’s disease

    Trends Neurosci.

    (1993)
  • R.E. Mrak et al.

    Glial cytokines in Alzheimer’s disease: review and pathogenic implications

    Hum. Pathol.

    (1995)
  • O. Ogawa et al.

    Inhibition of inducible nitric oxide synthase gene expression by indomethacin or ibuprofen in β-amyloid protein-stimulated J774 cells

    Eur. J. Pharmacol.

    (2000)
  • P.X. Petit et al.

    Mitochondria and programmed cell death: back to the future

    FEBS Lett.

    (1996)
  • C. Russo et al.

    Heterogeneity of water-soluble amyloid beta-peptide in Alzheimer’s disease and Down’s syndrome brains

    FEBS Lett.

    (1997)
  • T.C. Saido et al.

    Dominant and differential deposition of distinct β-amyloid peptide species, A beta N3(pE), in senile plaques

    Neuron

    (1995)
  • T.C. Saido et al.

    Amino- and carboxy-terminal heterogeneity of β-amyloid peptides deposited in human brain

    Neurosci. Lett.

    (1996)
  • D.J. Selkoe

    Amyloid beta-protein and the genetics of Alzheimer’s disease

    J. Biol. Chem.

    (1996)
  • G. Smale et al.

    Evidence for apoptotic cell death in Alzheimer’s disease

    Exp. Neurol.

    (1995)
  • T. Tomiyama et al.

    Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger

    J. Biol. Chem.

    (1996)
  • H.Y. Zhang et al.

    Huperzine B, a novel acetylcholinesterase inhibitor, attenuates hydrogen peroxide induced injury in PC12 cell

    Neurosci. Lett.

    (2000)
  • S.R. Abramson et al.

    The mechanisms of action of nonsteroidal antiinflammatory drugs

    Arthritis Rheum

    (1989)
  • D. Anrather et al.

    Thrombin activates nuclear factor-κB and potentiates endothelial cell activation by TNF

    J. Immunol.

    (1997)
  • M. Bisaglia et al.

    C3-fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis

    J. Neurochem.

    (2000)
  • R.C. Dodel et al.

    Sodium salicylate and 17β-estradiol attenuate nuclear transcription factor NF-κB translocation in cultured rat astroglial cultures following exposure to amyloid A β(1-40) and lipopolysaccharides

    J. Neurochem.

    (1999)
  • G. Emilien et al.

    Prospects for pharmacological intervention in Alzheimer disease

    Arch. Neurol.

    (2000)
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