Background: Oxidative stress plays a significant role in the physiological aging process and in some pathological conditions, such as Alzheimer's disease (AD). Studies suggested that biomarkers of oxidative and nitrosative stress are elevated in brain of AD patients and that oxidative damage is one of the earliest events in AD. In this experimental study, we focused on protein carbonylation and S- nitrosylation, which are irreversible and reversible post-translational modifications, respectively. The aim of this project was to identify protein markers of oxidative stress as an early event in AD pathogenesis. We hypothesized first that levels of carbonylated and nitrosylated membrane proteins of red blood cell (RBC) - including spectrin -, are increased in patients with cognitive impairment (CI) compared to controls, as based on measurements made in human autopsy brain tissue. The CI group includes patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). Secondly we split the CI group in MCI and AD groups, and hypothesized that AD contains less oxidized RBC proteins than MCI, supporting the idea that oxidative stress decreases with the disease progression.
Method: We analysed blood samples from a cohort of one hundred and twenty subjects aged 49 to 85 years, including 69 cognitive impairment (CI) subjects and 51 age-matched controls. The CI group was composed of MCI and AD patients. RBC membrane proteins were extracted and separated by one-dimensional electrophoresis. Fluorescent detection was then performed by using anti- dinitrophenyl (DNP) and anti-S-nitrocysteine antibodies to detect carbonylated and S-nitrosylated proteins, respectively. Then we quantified total RBC-membrane protein content and RBC-spectrin proteins, as well as their oxidation levels. Finally, we compared the levels of oxidation in CI and control groups. Further comparisons were performed in groups with/without established molecular biomarkers of AD risk, i.e. increased p-tau/Abeta1-42 ratio in cerebrospinal fluid (CSF) and APOE4 allele carriers. Two-dimensional gel-electrophoresis of selected RBC-membrane extracts was performed to test for differences in protein content and oxidation.
Results: The data revealed that protein carbonylation and S-nitrosylation were significantly decreased in cases of CI, as determined by a clinical dementia rate (CDR) score compared to controls. Oxidized and nitrosylated proteins also tended to decrease significantly in the group with pathological p-tau/Abeta1-42 ratio. Finally, S-nitrosylated beta spectrin was found decreased in APOEepsilon4 carriers when compared to APOEepsilon4 non-carriers. Two-dimensional gel-electrophoresis of selected RBC-membrane extracts revealed the presence of heatshock protein 27 (hsp27) and an elongation factor 2 (EF2) in CI samples.
Conclusion: The results showed that the blood of CI patients contained less oxidized proteins than controls. This discovery might suggest that oxidative stress has a role in the beginning or before the dementia development, and suggests an activation of stress response in the blood, possibly by activation of heatshock proteins. Oxidative damages in blood are not accumulated as it was found in autopsy brain tissue, but may be eliminated due to the RBC turnover and microvesiculation process, thus decreasing the levels of marker proteins. The relation between oxidative stress in blood and AD should be more studied by measuring the capacity of antioxidant mechanisms and following the progression of the oxidative protein pattern in the same patients. Such an understanding may be necessary in order to develop the diagnostic tools to evaluate the role of redox mechanisms in peripheral tissues as well as for targeting and designing antioxidant therapies.