Acrolein is increased in Alzheimer’s disease brain and is toxic to primary hippocampal cultures
Introduction
Increasing evidence supports the potential role of oxidative stress in neuronal degeneration in Alzheimer’s disease (AD). Studies show an increase in the redox active metal iron [6], [10], [41] as well as increased levels of lipid peroxidation [18], a decline in membrane polyunsaturated fatty acids [34], [42], and increased protein [9], [14] and DNA oxidation [8], [22], [29] in the brain in AD. Studies from our laboratory demonstrate increased levels of oxidatively modified DNA in AD ventricular cerebrospinal fluid (CSF) concomitant with decreased levels of the free repair product [21], and increased levels of 4-hydroxynonenal (HNE), a neurotoxic marker of lipid peroxidation, in AD brain [25] and ventricular CSF [19]. Elevated levels of F2-isoprostanes and F4-neuroprostanes, markers of lipid peroxidation, are found in AD CSF [31], [36], and increased levels of F4-isoprostanes are present in the AD brain [33]. Markers of oxidative stress in neurofibrillary tangles and senile plaques are present in the brain in AD [11], [30], [38], [39], [40], [46].
Peroxidation of lipids leads to the formation of a number of aldehydic by-products including malondialdehyde, C3–C10 straight chain aldehydes, and α,β-unsaturated aldehydes, such as HNE and acrolein [7], [37], [46].
Acrolein (CH2=CH-CHO) occurs in the environment as a ubiquitous pollutant that is generated as a by-product of overheated organic materials. In vivo, acrolein is formed in the metal-catalyzed oxidation of polyunsaturated fatty acids including arachidonic acid [43]. Acrolein is the strongest electrophile among the unsaturated aldehydes and shows the highest reactivity with nucleophiles including sulfhydryl groups of cysteine, histidine and lysine [7]. Acrolein formed in vivo through iron-catalyzed oxidation of arachidonic and docosahexenoic acids exhibits facile reactivity with various biomolecules including proteins and phospholipids, has the potential to inhibit many enzymes, and quickly depletes cellular glutathione levels [13]. It is postulated that acrolein may inactivate the reductase responsible for reducing vitamin E radicals [13] and, coupled with the depletion of glutathione, leads to further lipid peroxidation. Acrolein is capable of modifying DNA bases with the formation of exocyclic adducts [5], [26]. Studies of Uchida et al. [43], [44] and Esterbauer et al. [7] demonstrated that acrolein is rapidly incorporated into proteins and generates carbonyl derivatives. More recently, Uchida et al. [43] demonstrated that acrolein preferentially reacts with lysine residues that are prominent components of tau, and Calingasan et al. [3] described the presence of acrolein adducts in neurofibrillary tangles and dystrophic neurites surrounding senile plaques in AD.
This report demonstrates that acrolein is significantly elevated in the brain in AD compared with age-matched control subjects. Our results also indicate that acrolein is toxic to cultured hippocampal neurons in a time- and concentration-dependent manner and that the toxicity may be mediated through a disruption of calcium homeostasis.
Section snippets
Materials
Heptanal and propanal were from Waco Chemical (Japan). Acrolein, chloroform, 1,3 cyclohexanedione, methanol and tetrahydrofuran (THF) were from Aldrich Chemical (Milwaukee, WI, USA). Solid phase extraction columns, the C18 analytical HPLC column, and HPLC components were from Waters Corporation (Milford, MA, USA). Tissue culture reagents were from GibcoBRL, Life Technologies, Inc. (Rockville, NY, USA). The Pierce bicinchoninic acid (BCA) protein assay kit was from Pierce (Rockford, IL, USA).
Results
Representative HPLC chromatograms of AD and control HPG are shown in Fig. 1A and Fig. 1B. Identification of chromatographic peaks from tissue samples was based on comparison to a reference chromatogram of authentic acrolein (Fig. 1C). Quantification of acrolein concentration was based on comparison of acrolein peak area to the peak area of a known concentration (1.43 mM) of the internal standard (heptanal) peak area. Mean ± SEM brain acrolein concentrations are summarized in Fig. 2. Elevated
Discussion
This is the first study to quantitate an increase in the concentration of acrolein in vulnerable brain regions in AD patients compared with age-matched control subjects and to demonstrate a mechanism of acrolein-induced neuron death in vitro. Comparison of Figs. 1A and 1B demonstrates that the chromatographic profile for extracted aldehydes from brain specimens corresponds to the profile obtained for the reference standard, which consisted of derivatized free aldehydes. Because of acrolein’s
Acknowledgements
Funding for this work was provided by NIH Grants 5-P01-AG05119, and 5-P50-AG05144, and by a grant from the Abercrombie Foundation. The authors thank Paula Thomason for editorial and Jane Meara for technical assistance in manuscript preparation, and Cecil Runyons for demographic data. We also thank Dr. Robert Hadley and Hema Gursahani for assistance with quantification of intracellular calcium.
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