Electrochemical Analysis of Protein Nitrotyrosine and Dityrosine in the Alzheimer Brain Indicates Region-Specific Accumulation

Abstract

HPLC with electrochemical array detection (HPLC-ECD) was used to quantify 3,3Ј-dityrosine (diTyr) and 3-nitrotyrosine (3-NO 2 -Tyr) in four regions of the human brain that are differentially affected in Alzheimer's disease (AD). DiTyr and 3-NO 2 -Tyr levels were elevated consistently in the hippocampus and neocortical regions of the AD brain and in ventricular cerebrospinal fluid (VF), reaching quantities five-to eightfold greater than mean concentrations in brain and VF of cognitively normal subjects. Uric acid, a proposed peroxynitrite scavenger, was decreased globally in the AD brain and VF. The results suggest that AD pathogenesis may involve the activation of oxidant-producing inflammatory enzyme systems, including nitric oxide synthase.Key words: HPLC; nitrotyrosine; dityrosine; Alzheimer's disease; protein oxidation; inflammation The Alzheimer's disease (AD) brain exhibits region-specific patterns of amyloid plaque deposition, neurofibrillary tangle (NFT) accumulation, and neuron death. The limbic system and association areas of the neocortex show the most pronounced histopathological alterations in AD, whereas cortical somatosensory and cerebellar neurons are relatively spared (Pearson et al., 1985;Henderson and Finch, 1989;Braak and Braak, 1994). Recent models of AD attempt to link disease progression with an inflammatory component combined with increased oxidative stress (Rogers et al., 1996). C lassical hallmarks of inflammation such as edema and neutrophil infiltration are not acknowledged characteristics of the AD brain, although numerous correlates of inflammation are present. Acute-phase reactants such as C-reactive protein, major histocompatibility complex glycoproteins, complement, monocyte chemoattractants, interleukin-1, and interleukin-6 are elevated in AD brain in spatial association with neuritic plaques (Griffin et al., 1989McGeer et al., 1989;Bauer et al., 1991;Strauss et al., 1992;C arpenter et al., 1993; Wood et al., 1993;Iwamoto et al., 1994;Mrak et al., 1995;Pereira et al., 1996;Rogers et al., 1996;Sheng et al., 1996). Reactive microglia, f unctionally similar to monocytes, are increased in the AD brain and concentrate near senile plaques (McGeer et al., 1987;Haga et al., 1989;Itagaki et al., 1989; Carpenter et al., 1993;MacKenzie et al., 1995).Enhanced oxidative stress in the AD brain is manifested by increases in protein carbonyl content and lipid and DNA oxidation products and by inactivation of sensitive enzymes (Oliver et al., 1987;C. Smith et al., 1991C. Smith et al., , 1992Mecocci et al., 1993;Balazs and Leon, 1994;Chen et al., 1994;Hensley et al., 1995; Lovell et al., 1995;M. Smith et al., 1996;Butterfield et al., 1997;Lyras et al., 1997;Sayre et al., 1997). Correlation between oxidative and inflammatory biomarkers has not been achieved in the AD brain, although the activation of an inflammatory response might, in large part, explain AD brain oxidation. For instance, activated microglia release superoxide (O 2 ⅐ Ϫ ) and hydrogen peroxide (H 2 O 2 ) (Colton et al., 1994), whe...

Low expression of human tubulin tyrosine ligase and suppressed tubulin tyrosination/detyrosination cycle are associated with impaired neuronal differentiation in neuroblastomas with poor prognosis

“…Therefore, whether nitrotyrosinated tubulin is harmful or not is still controversial. Nevertheless, as increased nitrotyrosination is reported in Alzheimer's disease and amyotrophic lateral sclerosis, [35][36][37] the functional analysis of the role of hTTL and tubulin tyrosination/detyrosination cycle should be important for understanding the pathogenesis of these disease. The treatment of cells with methylmercury (MeHg) is also reported to induce perturbation of cellular activities associated with the tubulin/microtubule system by altering the status of tubulin tyrosination in the rat FIGURE 5 -Expression of hTTL mRNA is associated with unfavorable prognosis of neuroblastoma.…”

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease

“…Myeloperoxidase also oxidizes nitrite to reactive nitrogen species (Eiserich et al 1998;Byun et al 1999) and converts tyrosine to tyrosyl radical, a reactive intermediate that promotes o,o¢-dityrosine formation and initiates lipid peroxidation (Heinecke et al 1993;Savenkova et al 1994). This array of oxidatively modified amino acids bears a striking similarity to the markers of oxidative stress that are elevated in the brains of AD patients (Vitek et al 1994;Yan et al 1994;Sayre et al 1997;Hensley et al 1998;Pratico et al 1998).…”

“…Several markers of oxidative stress are increased in brain tissue from Alzheimer's patients, including advanced glycation end products (AGEs; Vitek et al 1994;Yan et al 1994), o,o¢-dityrosine (Hensley et al 1998), lipid oxidation products Pratico et al 1998), protein carbonyls (Smith et al 1998), oxidized DNA (Mecocci et al 1994;Gabbita et al 1998), and 3-nitrotyrosine . Oxidative damage occurs early in the neurodegenerative process (Nunomura et al 2001) and co-localization of oxidative stress markers with both neurofibrillary tangles (Good et al 1996;Sayre et al 1997;Smith et al 1997) and amyloid plaques (Smith et al 1994;Vitek et al 1994;Wong et al 2001;McLellan et al 2003) supports a role of oxidants in disease progression.…”

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

“…Protein nitration has been detected in patients suffering from a number of diseases such as Alzheimer's disease [13], atherosclerosis [14], amyotrophic lateral sclerosis [15], stroke [16], multiple sclerosis [17], cystic fibrosis [18], asthma [19] and diabetes [20]. However, the identification of nitrated proteins and their nitration sites has proved to be a challenging task.…”