Multiple studies have demonstrated elevations of α, β-unsaturated aldehydes including 4-hydroxynonenal (HNE) and acrolein, in vulnerable regions of mild cognitive impairment (MCI), preclinical Alzheimer's disease (PCAD), and late stage Alzheimer's disease (LAD) brain. However, there has been limited study of a third member, 4-hydroxyhexenal (HHE), a diffusible lipid peroxidation product of the ω-3 polyunstataturated fatty acids (PUFAs). In the present study levels of extractable and protein-bound HHE were quantified in the hippocampus/parahippocampal gyrus (HPG), superior and middle temporal gyri (SMTG), and cerebellum (CER) of MCI, PCAD, LAD, and normal control (NC) subjects. Levels of extractable and protein-bound HHE were increased in multiple regions in the progression of Alzheimer's disease (AD). Extractable HHE was significantly elevated in the hippocampus/parahippocampal gyrus (HPG) of PCAD and LAD subjects and protein-bound HHE was significantly higher in MCI, PCAD, and LAD HPG. A time- and concentration-dependent decrease in survival and a concentration-dependent decrease in glucose uptake were observed in primary cortical cultures treated with HHE. Together these data support a role for lipid peroxidation in the progression of Alzheimer's disease.
Oxidative damage to cellular macromolecules, including nucleic acids, proteins, and lipids, is a feature of aging as well as many neurodegenerative diseases including Alzheimer's disease (AD). Multiple studies have shown oxidative damage, including nucleic acid oxidation (Gabbit et al., 1998, Lovell et al., 1999, Mecocci et al., 1994, Nunomura et al., 2001 and Shan et al., 2003), protein modifications (Ding et al., 2006, Drake et al., 2004, Lovell et al., 1998 and Lovell et al., 2000b; Pocernich and Butterfield, 2003, Shao et al., 2008 and Sultana et al., 2006), and generation of by-products of lipid peroxidation (Lovell et al., 2001, Markesbery and Lovell, 1998 and McGarth et al., 2001) are significantly increased in late-stage Alzheimer's disease (LAD) compared with age-matched normal control (NC) subjects. In addition, markers of oxidative damage have been observed in mild cognitive impairment (MCI), the earliest clinical manifestation of AD (Butterfield et al., 2006, Ding et al., 2005, Markesbery et al., 2005, Shao et al., 2008, Wang et al., 2006 and Williams et al., 2006). These observations suggest oxidative damage may play a potential role in the pathogenesis of AD.
Lipids represent a class of biomacromolecules whose proper function is vital to cellular homeostasis, but are also vulnerable to oxidative damage by reactive oxygen species (ROS). Peroxidation of lipids results in compromised integrity of cellular membranes and the generation of diffusible aldehydic by-products including the α, β-unsaturated aldehydes acrolein, 4-hydroxyhexenal (HHE), and 4-hydroxynonenal (HNE). The toxicity of α, β-unsaturated aldehydes is attributed to their soft electrophilic nature that is highly reactive with cysteine, histidine, and lysine amino acid residues (LoPachin et al., 2009). HNE and acrolein are elevated in MCI and LAD brain (Butterfield et al., 2006, Lovell et al., 2001, Markesbery and Lovell, 1998, Reed et al., 2008 and Williams et al., 2006) and have been shown to be toxic in neuron cultures (Lovell et al., 2000a, Lovell et al., 2001 and Pocernich and Butterfield, 2003). HHE is a by-product of oxidative damage to ω-3 polyunsaturated fatty acids (PUFAs) including docosahexaenoic acid, the predominate ω-3 PUFA in gray matter (Long et al., 2008 and Van Kuijk et al., 1990). Concentrations of docosahexaenoic acid (DHA) are approximately 30–50 times higher than the predominate ω-6 PUFA, arachidonic acid (Lim et al., 2005, Pawlosky et al., 2001 and Salem et al., 2001) making it an abundant target for oxidative attack.
In the current studies levels of extractable and protein-bound HHE were quantified in vulnerable brain regions, the hippocampus/parahippocampal gyrus (HPG) and superior and middle temporal gyri (SMTG), and the cerebellum (CER), a nonvulnerable brain region from preclinical Alzheimer's disease (PCAD), MCI, LAD, and NC subjects. Levels of extractable HHE were quantified using gas chromatography mass spectrometry with negative chemical ionization (GC/MS/NCI) and protein-bound levels of HHE were determined by dot blot immunochemistry and an HHE specific antibody. In addition, levels of protein carbonyls were quantified by immunochemistry. To determine the rate of HHE generation in the presence of AD physiologically relevant oxidative insults, DHA was treated with amyloid β peptide (Aβ1–40, Aβ1–42), as well as iron (II)/ascorbic acid. Levels of extractable HHE generated from oxidized DHA were determined by gas chromatography mass spectrometry with negative chemical ionization. To investigate the effect of HHE on primary cortical neurons, cell viability was assessed at 3, 6, 12, and 24 hours in cultures treated with increasing HHE concentrations (1–100 μM). Additionally, the effect of HHE (1 to 100 μM) on glucose uptake was assessed at 6 hours.
