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Butterfield 20et 20al 20 2007 20Free 20Radical 20Biology 20 20Medicine 2043 20658 677 Powered By Docstoc
					                                                                 Free Radical Biology & Medicine 43 (2007) 658 – 677
                                                                                                                                                                                                 www.elsevier.com/locate/freeradbiomed


                                                                                                 Review Article
   Roles of amyloid β-peptide-associated oxidative stress and brain protein
        modifications in the pathogenesis of Alzheimer's disease and
                          mild cognitive impairment
                   D. Allan Butterfield ⁎, Tanea Reed, Shelley F. Newman, Rukhsana Sultana
                                         Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA
                                      Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536, USA
                                       Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506, USA
                                                 Received 31 March 2007; revised 20 May 2007; accepted 25 May 2007
                                                                   Available online 13 June 2007



Abstract

   Oxidative stress has been implicated to play a crucial role in the pathogenesis of a number of diseases, including neurodegenerative disorders,
cancer, and ischemia, just to name a few. Alzheimer disease (AD) is an age-related neurodegenerative disorder that is recognized as the most
common form of dementia. AD is histopathologically characterized by the presence of extracellular amyloid plaques, intracellular neurofibrillary
tangles, the presence of oligomers of amyloid β-peptide (Aβ), and synapse loss. In this review we discuss the role of Aβ in the pathogenesis of
AD and also the use of redox proteomics to identify oxidatively modified brain proteins in AD and mild cognitive impairment. In addition, redox
proteomics studies in in vivo models of AD centered around human Aβ(1–42) are discussed.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Alzheimer's disease; Mild cognitive impairment; Oxidative stress; Amyloid β-peptide; Redox proteomics; Free radicals




Contents

   Introduction . . . . . . . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   659
   Amyloid β-peptide . . . . .       .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   661
   Oxidative stress in AD brain      .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   663
       Protein oxidation . . . .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   663
       Protein nitration . . . . .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   663


    Abbreviations: 3-NT, 3-nitrotyrosine; 8-OHdG, 8-hydroxy-2-deoxyguanine; 8-OHG, 8-hydroxyguanine; Aβ, amyloid β-peptide; AD, Alzheimer disease; APP,
amyloid precursor protein; CAII, carbonic anhydrase II; CK BB, creatine kinase BB isoform; CSF, cerebrospinal fluid; DHAP, dihydroxyacetone phosphate; DNPH,
2,4-dinitrophenylhydrazine; DRP2, dihydropyrimidinase-related protein 2; ESI, electrospray ionization; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde-
3-phosphate dehydrogenase; GS, glutamine synthase; GST, glutathione S-transferase; HNCP, hippocampal cholinergic neurostimulating protein; HNE, 4-hydroxy-2-
trans-nonenal; HSC 71, heat shock cognate 71; HSP60, chaperonin 60; ICAT, isotopically coded affinity tags; IEF, isoelectric focusing; iNOS, inducible nitric oxide
                                                                     U
synthase; IPL, inferior parietal lobe; LDH, L-lactate dehydrogenase; MALDI, matrix-assisted laser desorption/ionization; MCI, mild cognitive impairment; mtDNA,
mitochondrial DNA; MetO, methionine sulfoxide; MetS +, sulfuranyl radical; MPTP, mitochondrial permeability transition pore; MRP-1, multidrug-resistant protein;
                                                                                                                                                                                                                                                             U
                                                             U
NBM, nucleus basalis magnocellarius; nDNA, nuclear DNA; NFT, neurofibrillary tangles; NMDA, N-methyl-D-aspartic acid; nNOS, neuronal NOS; NO , nitric
oxide; NSF, N-ethylmaleimide-sensitive factor; O 2 −, superoxide radical anion; ONOO−, peroxynitrite; PEBP, phosphatidylethanolamine-binding protein; PGM1,
phosphoglycerate mutase 1; Pin1, peptidylprolyl-cis,trans isomerase 1; RNS, reactive nitrogen species; ROS, reactive oxygen species; γ-SNAP, γ-soluble
N-ethylmaleimide-sensitive attachment protein; SP, senile plaques; TBARS, thiobarbituric acid-reactive substance; TPI, triosephosphate isomerase; UCHL-1, ubiquitin
carboxy-terminal hydrolase L-1; VDAC, voltage-dependent anion channel protein.
  ⁎ Corresponding author. Fax: +1 859 257 5876.
    E-mail address: dabcns@uky.edu (D.A. Butterfield).

0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2007.05.037
                                     D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677                                                                   659


     Lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   663
     DNA oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   663
     RNA oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   664
     Mitochondrial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   664
     Vascular factors in the conversion from MCI to AD . . . . . . . . . . . . . . . . . . . . . .        . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   664
  Oxidative stress in MCI brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   665
  Redox proteomics identification of oxidatively modified brain proteins in AD . . . . . . . . . .        . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   665
     Redox proteomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   665
     Mass spectrometry and database searching . . . . . . . . . . . . . . . . . . . . . . . . . . .       . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   666
     Oxidative modification of brain proteins in AD: carbonylation and nitration . . . . . . . . .        . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   666
     Functional classification of redox proteomics-identified oxidatively modified brain proteins in      AD .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   667
          Energy-related enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   667
          Proteasome-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   667
          Structural proteins: β-actin and DRP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . .    . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   668
          Cholinergic function and lipid asymmetry . . . . . . . . . . . . . . . . . . . . . . . . .      . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   668
          Neurotransmitter-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   668
          Synaptic abnormalities and long-term potentiation . . . . . . . . . . . . . . . . . . . . .     . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   668
          pH regulation protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   668
          Cell cycle, tau phosphorylation, Aβ production . . . . . . . . . . . . . . . . . . . . . .      . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   668
          Mitochondrial-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   669
  Redox proteomics: identification of oxidatively modified brain proteins in MCI. . . . . . . . . .       . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   669
  Redox proteomics analysis of in vivo models of AD centered around human Aβ(1–42) . . . . .              . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   669
     Human Aβ(1–42) injected into rat cholinergic-rich basal forebrain . . . . . . . . . . . . . .        . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   669
     Human Aβ(1–42)-expressing Caenorhabditis elegans . . . . . . . . . . . . . . . . . . . . .           . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   669
     Canine model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   670
  Future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   670
  Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .       . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   670
  References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   670




Introduction                                                                 DNA and RNA oxidation. Among the earliest of these changes
                                                                             after an oxidative insult are increased levels of toxic carbonyls,
    Oxidative stress has been implicated to play a crucial role in           3-nitrotyrosine (3-NT), and HNE [2,4,7,10–13].
the pathogenesis of a number of diseases, including neurode-                     Protein carbonyl groups are generated by direct oxidation
generative disorders, cancer, and ischemia [1]. Among all the                of certain amino acid side chains (i.e., Lys, Arg, Pro, Thr, and
body organs, the brain is particularly vulnerable to oxidative               His); peptide backbone scission; Michael addition reactions of
damage because of its high utilization of oxygen, increased                  His, Lys, and Cys residues with products of lipid peroxidation;
levels of polyunsaturated fatty acid (that are readily attacked by           or glycol oxidation of Lys amino groups [6,14–17]. Protein
free radicals), and relatively high levels of redox transition               carbonyls are stable and hence are widely used as markers to
metal ions; in addition, the brain has relatively low levels of              assess the extent of oxidation of proteins under both in vivo
antioxidants [2–5]. The presence of iron ion in an oxygen-rich               and in vitro conditions [14,15,17,18]. The levels of protein
environment can further lead to enhanced production of                       carbonyls can be determined experimentally by derivatization
hydroxyl free radicals and ultimately lead to a cascade of                   of the carbonyl groups with 2,4-dinitrophenylhydrazine
oxidative events.                                                            (DNPH), followed by spectroscopic or immunochemical de-
    Oxidative stress occurs due to an imbalance in the pro-                  tection of the resulting hydrazone product [6,15,19].
oxidant and antioxidant levels. Reactive oxygen species (ROS)                    In addition to direct effects, oxidative stress could also
and reactive nitrogen species (RNS) are highly reactive with                 stimulate additional damage in brain via the overexpression of
biomolecules, including proteins, lipids, carbohydrate, DNA,                 inducible nitric oxide synthase (iNOS) and the action of
and RNA [6]. Oxidative damage to these moieties leads to                     constitutive neuronal NOS (nNOS) that increase the production
cellular dysfunctions [1,2,5,7–9]. The markers of oxidative
                                                                                                  U
                                                                             of nitric oxide (NO ) via the catalytic conversion of arginine to
stress that are commonly used in biological samples include
                                                                                                                                        U
                                                                             citrulline. Nitric oxide reacts with superoxide anion (O2 −) at a
protein carbonyls and 3-nitrotyrosine for protein oxidation;                 diffusion-controlled rate to produce peroxynitrite (ONOO−).
thiobarbituric acid-reactive substances (TBARS), free fatty acid             Peroxynitrite is highly reactive, with a half-life of less than a
release, iso- and neuroprostane formation, 2-propen-1-al                     second, and can undergo a variety of chemical reactions
(acrolein), and 4-hydroxy-2-trans-nonenal (HNE) for lipid                    depending upon its cellular environment, the presence of CO2,
peroxidation; advanced glycation end products for carbohy-                   and the availability of reactive targets forming modifications
drates; 8-OH-2′-deoxyguanosine and 8-OH-guanosine and                        such as 3-NT (Fig. 1) [20,21]. 3-Nitrotyrosine is a covalent
other oxidized bases, and altered DNA repair mechanisms for                  protein modification that has been used as a marker of nitro-
660                                 D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677




                     Fig. 1. Tyrosine nitration. (A) Reaction of peroxynitrite and carbon dioxide. (B) Formation of 3-nitrotyrosine.



sative stress in a variety of disease conditions [22,23]. Peroxy-              datively modified proteins may disrupt cellular functions by
nitrite can also react with sulfhydryl compounds intracellu-                   alterations in protein expression and gene regulation, protein
larly due to the high concentration of free thiols within the                  turnover, modulation of cell signaling, induction of apop-
cell [24]. Sulfhydryls can also react via S-nitrosylation with                 tosis and necrosis, etc., which suggests that protein oxidation
    U
NO to form a nitrosothiol. Cysteine residues are preferen-                     could have both physiological and pathological significance
tially nitrosylated due to favorable reaction kinetics [25,26].                [6,29–31]. In our laboratory, we used redox proteomics
    Protein oxidation could lead to aggregation or dimerization                analyses to identify specific oxidatively modified brain pro-
of proteins; in addition, protein oxidation can also lead to                   teins, carbonylated proteins, in neurodegenerative diseases and
unfolding or conformational changes in the protein, thereby                    models thereof [10,12,13,32–41].
exposing more hydrophobic residues to an aqueous environ-                         Mild cognitive impairment (MCI) is considered an inter-
ment. This exposure may lead to a loss of structural or                        mediate phase between normal aging and Alzheimer disease.
functional activity and protein aggregation and subsequent                     Some researchers believe that MCI is in fact the earliest form of
accumulation of the oxidized proteins as cytoplasmic inclu-                    AD [42]. Persons with MCI have cognitive complaint, decline
sions, such as tau aggregation in the form of tangles and                      in cognition compared to previous years, and, most notably, no
amyloid-β aggregation as senile plaques, as observed in                        signs of dementia. Additionally, activities of daily living are not
Alzheimer disease (AD) [27,28]. The accumulation of oxi-                       affected. These characteristics require informant confirmation
                                   D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677                             661


for diagnosis of MCI. Accurate diagnosis can be confirmed only              consequently, to early onset of AD [49]. Presenilin-1 and pre-
by medical examination to establish a level of cognitive decline            senilin-2 comprise the catalytic element of γ-secretase [54].
[43–45]. Pathologically, MCI has also been characterized using              Another critical component of γ-secretase is presenilin enhancer
magnetic resonance imaging technology to show measurable                    2 (Pen-2). Pen-2 binds to presenilins and subsequently enhances
atrophy in the hippocampus and entorhinal cortex [46,47].                   γ-secretase activity, thereby accumulating high levels of toxic
Alzheimer disease patients have considerable neurodegenera-                 Aβ(1–42) [55,56].
tion in these aforementioned areas. Because the hippocampus is                  Aβ(1–42) has a critical methionine residue at position 35,
the region of the brain primarily responsible for processing of             which is believed to be associated with the toxicity of Aβ [57–
memory, atrophy in this brain region is consistent with memory              61]. Oxidation of the Met35 produces methionine sulfoxide
loss in AD and MCI.                                                         (MetO) or, through further irreversible oxidation, methionine
   An age-related neurodegenerative disorder, AD is recognized              sulfone (Fig. 2) [62]. Methionine sulfoxide can be reduced back
as the most common form of dementia. AD is clinically                       to methionine by the enzyme methionine sulfoxide reductase.
associated with cognitive impairment, loss of language and                  This stereospecific reaction is facilitated by thioredoxin and is
motor skills, and changes in behavior. AD is pathologically                 NADPH-driven [63]. Methionine sulfoxide modulates oxida-
characterized by the presence of extracellular senile plaques,              tive stress and neurotoxic properties of Aβ(1–42), and studies
which consist of a core of Aβ, and intracellular neurofibrillary            have shown that the activity of methionine sulfoxide reductase
tangles (NFTs), and loss of synaptic connections within                     is reduced in AD brain [64,65].
entorhinal cortex and progressing into the hippocampus and                      This Met35 residue has been shown to be critical for
cortex. NFTs are composed of paired helical filaments that                  Aβ(1–42) toxicity and oxidative stress [57]. The secondary
consist of aggregates of the hyperphosphorylated microtubule-               structure of Aβ(1–42), which is helical as a small oligomer in
associated protein tau [48], whereas senile plaques (SPs) are rich          membrane lipid bilayers, contributes to the oxidative stress
in Aβ [49].                                                                 and neurotoxicity of this peptide [66]. When Ile31 of Aβ(1–42)
   In this paper, we review the involvement of Aβ and other                 is substituted by the helix-breaking amino acid proline, in the
sources of oxidative stress in AD brain and the use of redox                “i + 4” α-helix conformation, no protein oxidation occurs [66].
proteomics to identify oxidatively modified brain proteins in
                                                                                                        U
                                                                            A sulfuranyl radical (MetS +) results as the lone pair of electrons
AD and MCI. New insights into potential oxidative mechanisms                on the S atom undergoes a one-electron oxidation (Fig. 3)
underlying molecular processes in these disorders and progres-              [57,67,68]. The sulfuranyl radical can initiate free radical chain
sion from MCI to AD have emerged.                                           reactions with allylic H atoms on unsaturated acyl chains of
                                                                            lipids until a termination step is reached. Sulfuranyl radicals can
Amyloid β-peptide                                                           react with molecular oxygen to produce sulfoxide and super-
                                                                                                           U
                                                                            oxide [69]. Similarly, MetS + can react with oxygen to form
   Aβ, a 40- to 42-amino-acid peptide, is derived by proteolytic            MetO [70], making various proteins inactive [71]. Aβ(1–40) is a
cleavage of an integral membrane protein known as amyloid                   shorter peptide fragment produced from APP, which is also
precursor protein (APP) by the action of β- and γ-secretases.               neurotoxic [72]. There is an abundance of Aβ(1–40) MetO in
Aβ(1-40) and Aβ(1-42) constitute the majority of the Aβ found               senile plaques [73]. The oxidation of this methionine is reported
in human brain and have been considered to play a role in the               to prevent Aβ(1–40) from forming β sheets during fibril
development and progression of AD [49]. Aβ(1-42) is the more                formation [74]; therefore, Aβ(1–42) could be more toxic by this
toxic of these species both in vitro and in vivo. Further, a                mechanism. With the I31P substitution of Aβ(1–42), the
number of studies suggest that small oligomers of Aβ are                    interaction of the carboxyl oxygen of Pro31 with the S atom of
the actual toxic species of this peptide, rather than Aβ fibrils            Met35 is disrupted, yielding no neurotoxicity or protein
[50–53]. In addition, genetic mutations in genes for APP or                 oxidation in neurons [66]. The sulfuranyl radical can react
presenilin-1 or presenilin-2, which lead to familial AD, have               with the methylene moieties of Met to form the α-(alkylthio)
been reported to increase the production of Aβ(1-42) and lead,              alkyl radical of methionine (–CH2–CH2–S–CH2). Others




                               Fig. 2. Methionine chemistry. Formation of methione sulfoxide and methionine sulfone.
662                                        D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677




Fig. 3. Aβ(1–42) as a small oligomer is postulated to reside in the lipid bilayer. One-electron oxidation of the S atom on Met35, facilitated by the α-helical i +4
interaction of the backbone carbonyl of Ile31 with the S atom of Met35, forms a positively charged sulfuranyl radical that is stabilized in part by the helical dipole of
Aβ(1–42) located in the lipid bilayer. This radical can abstract a lipid acyl allylic H atom, forming a lipid carbon radical that immediately binds paramagnetic O2. This
peroxyl radical can abstract an allylic H atom, making the lipid hydroperoxide and propagating the chain reaction. The lipid hydroperoxide on arachidonic acid can
decompose to HNE, which can subsequently bind to proteins by Michael addition, resulting in oxidative damage to the protein. The SH+ acid on Met has a pKa of − 5,
so loses the H+ easily to reform Met. That is, the reaction is catalytic. This mechanism is consistent with an amplification of damage by a relatively minor degree of
conversion of Met to the sulfuranyl free radical of Aβ(1–42).


showed, using computer modeling, that the β-sheet conforma-                               If the Gly33 residue is substituted by the hydrophobic amino
tion of this toxic peptide contains the sulfur-centered radical                        acid valine, neurotoxicity is minimal and protein oxidation is
cation of Met35, which bolsters the creation of an α-carbon-                           significantly reduced in comparison to the wild-type Aβ(1–42)
centered radical on the Gly33 residue. This gives rise to a                            peptide [77]. Several groups [58,59,61] have confirmed our
hydrophobic environment that is ideal for lipid peroxidation in                        findings that substitution of the S atom of Met35 by CH2 (to
the lipid bilayer [75,76]. The sulfuranyl radical (a positively                        make the R group norleucine, Nle) abrogates the oxidative
charged radical) on the Met35 can in principle be stabilized by                        stress and neurotoxicity of the resulting peptide [68]. Other
the helix-associated dipole of the C-terminal end of Aβ(1–42).                         researchers point to the role of Tyr10 as a source of free radicals
Because the dipole of Aβ(1–42) would be greater than that of                           of Aβ(1–42). This free radical is proposed to reduce Cu2+
                                                                                       bound to His6, His13, and His14, leading to Cu+-mediated
Aβ(1–40), the differential toxicity of Aβ(1–42) and Aβ(1–40)
may be related to this characteristic. The importance of the
                                                                                                                  U
                                                                                       chemistry to produce OH [78,79]. These researchers point out
hydrophobic environment of the Met35 residue was evaluated.                            that rat Aβ(1–42) lacks Tyr10 and is not toxic. However,
By substituting aspartic acid for Gly37, the critical methionine                       substitution of Tyr10 by Phe (to keep aromaticity present but to
residue no longer remained in a hydrophobic environment, and                           prevent any possible electron flow in contrast to this possibility
Aβ(1–42) oxidative stress and neurotoxicity were abrogated                             with Tyr), as well as substitution of three His residues by Asn
[76]. This result suggests that Aβ(1–42)-mediated lipid                                (which binds Cu2+ at least 100-fold less than His), led to a toxic
peroxidation is an early and crucial step in the toxicity of this                      peptide similar to native human Aβ(1–42) [57]. Also, rat
peptide.                                                                               Aβ(1–42) was shown to be toxic upon longer incubation with
                                   D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677                           663


neurons [80]. The p3 fragment of human Aβ(1–42), Aβ(17–                    notion that nitrosative stress also contributes to neurodegen-
42), which does not contain Tyr10 or His6, His13, and His14,               eration in AD [10,11,13,16,86]. Protein nitration also in-
was reported to be toxic [57]. This Met-containing peptide,                creases the susceptibility of brain proteins to proteasomal
which is present in AD brain, was no longer toxic if the Met35             degradation [26]. The overexpression of iNOS and nNOS
was substituted by Nle [57]. The dissociation constant of Cu2+             could be responsible for increased levels of RNS. Previous
binding to Aβ(1–42) was reported to be attomolar [79].                     studies have reported mitochondrial abnormalities in the AD
Whereas some of these researchers propose an AD therapy
                                                                                                              U
                                                                           brain [87], which could lead to O2 − leakage. As shown in Fig. 1,
based on Cu2+ chelation using clioquinol, which has a Kd that is           superoxide then reacts with NO
                                                                                                             U to form ONOO−.
nanomolar [81], the 9 orders of magnitude difference in affinity               Tyrosine is particularly susceptible to nitration but
for Cu2+ between Aβ(1–42) and clioquinol make it unlikely                  tryptophan and phenylalanine can also be nitrated, albeit at
that the effectiveness of this agent is correctly ascribed to its          lower rates. Tyrosine residues are important to redox cell
chelating ability. However, we propose that clioquinol could               signaling and are often the site of phosphorylation, a
chelate relevant weakly bound Cu2+, for example, if Cu2+                   prominent regulation function [88]. The nitration of tyrosine
were loosely bound to Met35 of Aβ(1–42). This scenario                     at the 3-position sterically hinders the phosphorylation and
would account for the oxidation of Met to a sulfuranyl radical,            also may change the structure of proteins, thereby rendering a
whereas Cu2+ would be reduced to Cu+, whence further                       protein dysfunctional, and could lead to cell death [6,89].
oxidative chemistry conceivably could ensue. Further inves-                    Increased levels of nitrated proteins have been reported to be
tigation will be required to test this notion. Thus, whereas a             present in AD brain and cerebrospinal fluid (CSF), implying a
role for Cu2+/Cu+ in the oxidative stress and neurotoxicity of             role for RNS in AD pathology [10,11,13,90]. An increase in 3-
Aβ(1–42) cannot be excluded, it seems that there is a paramount            NT immunoreactivity in neurons from AD brain was observed
role for Met35 in these properties.                                        [11], as well as elevated dityrosine and 3-NT levels in
   Finally, several researchers employ the 11-mer, Aβ(25–35),              hippocampus, inferior parietal lobule (IPL), and neocortical
as a model for the AD-relevant (but more costly) Aβ(1–42).                 regions of the AD brain and in ventricular cerebrospinal fluid
Even in the case of the 11-mer, substitution of the C-terminal             compared with aged matched controls [11,86]. The demonstra-
Met by Nle abrogated the free radical oxidative stress induced             tion of nitrated tau in pretangles, tangles, and tau inclusions in
by this shorter peptide as did use of the 10-mer Aβ(25–34) [68].           AD brain suggests that tau nitration may be an early event in AD
Moreover, because Met is C-terminal in Aβ(25–35), whereas                  [91]. This notion is strengthened by our finding of elevated 3-NT
Met is intrachain in Aβ(1–42), the mechanism by which the                  in the brains of subjects with MCI (see below).
sulfuranyl free radical on Met35 is generated is different in these
two peptides [68]. This fact, coupled with the absence of                  Lipid peroxidation
evidence for Aβ(25–35) in AD brain, suggests to us that use of
Aβ(25–35), though of possible academic interest, is of no                     Amplified lipid peroxidation has been described in several
relevance for gaining insight into AD.                                     neurodegenerative diseases, including AD [7,83,84,92]. Analy-
                                                                           sis of AD brains demonstrates an increase in free HNE in
Oxidative stress in AD brain                                               amygdala, hippocampus, and parahippocampal gyrus of the AD
                                                                           brain compared with age-matched controls [83]. This increased
   Oxidative stress is observed in the AD brain [1–5]. This                alkenal concentration corresponds with the regions showing the
increase has been well documented with markers for protein,                most striking histopathologic alterations in AD. A significant
DNA, and RNA oxidation as well as lipid peroxidation                       elevation of free HNE in ventricular CSF and serum provides a
[4,6,11,82,83].                                                            potential biomarker for AD [93,94]. Protein-bound HNE, which
                                                                           is an indication of Michael addition of HNE to proteins, is
Protein oxidation                                                          elevated in AD [2,4,84,95]. HNE is elevated in neurons treated
                                                                           with Aβ(1–42) [8,84,96]. Protein-bound HNE alters conforma-
   Protein oxidation is indexed in the AD brain by an increase             tion and function of proteins [84,97]. In AD brain protein-bound
in carbonylated [82] and HNE- [84] and 3-NT- [11] modified                 HNE is found on the glutamate transporter, glutathione S-
proteins. The initial origin of AD pathogenesis has not been               transferase (GST), and multidrug-resistant protein (MRP-1)
determined though it has become evident that oxidative stress is           [84,98]. Thus processes related to excitotoxicity may be
implicated in the development of this disease [1,5,85]. Studies            facilitated, whereas processes related to the removal of HNE
have shown an increase in protein carbonyls in the hippocampus             from neurons (via GST and MRP-1) may be compromised.
and parietal cortex, but not in the cerebellum, where there is less
significant AD pathology [82].                                             DNA oxidation

Protein nitration                                                             More than 4 decades ago, it was suggested that DNA is the
                                                                           primary target of ROS, leading to cellular aging [99]. Due to the
   Another common marker of protein oxidation is the                       high oxygen consumption rate by the brain, ROS may contri-
addition of a nitro group to tyrosine residues forming 3-NT.               bute to neuronal damage in aging and neurological disorders
Increased protein nitration in the AD brain supports the                   [5]. Oxidative damage to DNA by ROS results in strand breaks,
664                                D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677


DNA–DNA and DNA–protein cross-linking, and sister-chro-                    α-ketoglutarate dehydrogenase activity in the parietal and
matid exchange and translocation [100,101]. DNA bases are                  temporal cortex of AD patients has also been reported,
also attacked by the lipid peroxidation products HNE and                   which could be related to the HNE binding to α-ketoglutarate
acrolein, which leads to the formation of bulky exocyclic                  [126–130], though this could be affected by longer postmortem
adducts. This modification can cause inappropriate base pairing            intervals [131]. All the above-mentioned mitochondrial changes
that alters protein synthesis. DNA oxidation by ROS also pro-              would limit ATP production and increase ROS production and
duces oxidized base adducts, such as 8-hydroxy-2-deoxygua-                 suggest possible abnormalities in mitochondrial functions in
nine (8-OHdG) [102,103]. Guanine, because it has the lowest                AD. ROS production is directly related to the mitochondrial
oxidation potential of the four DNA bases, is the most readily             membrane potential (ΔΨ) such that hyperpolarization (high
oxidized base and therefore the most commonly used analysis                ΔΨ) promotes ROS production [132–134]. In addition, several
of DNA oxidation.                                                          in vitro studies of Aβ and mitochondrial function have reported
   Previous studies have demonstrated a 2-fold increase in                 that Aβ affects mitochondrial DNA and proteins, leading to
DNA strand breaks in AD brain that consequently results in                 impairment of the ETC and ultimately mitochondrial dysfunc-
depletion of energy stores and cell death [104]. DNA oxidation             tion [135,136]. Dysfunction of mitochondria is reported to alter
has been shown to escalate with age, shown by an increase in               APP metabolism, enhancing intraneuronal accumulation of
8-OHdG in the cerebral cortex and cerebellum brain regions                 amyloid β-peptide and enhancing neuronal vulnerability [137].
[105]. Similarly both mitochondrial DNA (mtDNA) and                        Consistent with the importance of Aβ(1–42) in AD pathogen-
nuclear DNA (nDNA) have increased 8-hydroxyguanine (8-                     esis, recent research has suggested the presence of intracellular
OHG), 8-hydroxyadenine, and 5-hydroxyuracil in temporal,                   Aβ(1–42) in mitochondria from brains of transgenic mice and
parietal, and frontal lobes in AD [105,106]. Overall, mtDNA                AD patients [138,139], and showed increased expression of
shows an approximately 10-fold higher intensity of oxidized                mitochondrial genes before Aβ deposition in APP transgenic
bases than nDNA. This result demonstrates that nDNA and                    mice, and suggested that the upregulation of mitochondrial
mtDNA undergo extensive oxidative damage in AD, which                      genes may be a compensatory response because of mitochon-
may contribute to the neurodegenerative pathology of this                  drial oxidative damage caused by the overexpression of mutant
disorder.                                                                  APP and/or Aβ [139]. Further, the accumulation of Aβ in the
                                                                           mitochondria may be associated with diminished enzymatic
RNA oxidation                                                              activity of mitochondria [140], where the peptide is proposed to
                                                                           disrupt energy production while promoting mitochondrially
    Studies have shown 30–70% oxidation of the mRNAs in the                derived apoptotic processes via the intrinsic pathway. This claim
frontal cortex of the AD brain in comparison to only 2%                    of mitochondrial Aβ needs replication but is a provocative
oxidation in age-matched controls [107]. A specific increase in            hypothesis that provides potential insight into metabolic
rRNA oxidation has also been shown in the AD IPL compared                  alterations and oxidative stress in AD brain.
to age-matched controls [108]. Increased levels of 8-OHG have
also been reported in the hippocampus and cerebral neocortex               Vascular factors in the conversion from MCI to AD
of the AD brain, whereas the 8-OHG level in the cerebellum
was not significantly altered compared with controls [108–110].                Oxidative stress is also associated with vascular factors
An increase in 8-OHdG has been identified not only in brain                [141–144] that reportedly play a role in the conversion of MCI
tissue but also in CSF from AD patients [111]. RNA oxidation               to AD [145]. Vascular factors include, but are not limited to,
in the AD brain could render the cell incapable of initiating              ApoE4 allele, diabetes, smoking, hypertension, and heart
protein synthesis, hindering the cell's defense against further            disease [146]. The ApoE4 isoform is associated with increased
oxidative damage, an effect observed in AD [108].                          peripheral lipid levels, glial cell activation, decreased peripheral
                                                                           lipid metabolism, and cerebral glucose metabolism, which may
Mitochondrial dysfunction                                                  eventually lead to inflammation, oxidative stress, and dementia.
                                                                           A recent study by Wolozin and Bednar [147] suggests that
   Mitochondrial dysfunction in AD is central to the develop-              ApoE does not directly contribute to AD by increasing serum
ment of oxidative stress because it is a primary source of cellular        cholesterol, but rather ApoE might act via a mechanism
oxidants [112,113]. In vivo positron emission tomography                   involving Aβ [147]. In our laboratory we showed that increased
(PET) has provided specific evidence of brain metabolism                   oxidative stress in APP/PS-1 mice is independent of dietary
abnormalities associated with AD [114], which precede                      cholesterol, which further supports the role of Aβ in the
neuropsychological impairment and visual atrophy [115,116].                induction of oxidative stress rather than the direct effect of
Frontal cortex and middle temporal gyrus show a significant                cholesterol [148]. In addition to ApoE other risk factors for AD
decrease in metabolism as well as synaptic dysfunction [117–               are coronary artery bypass surgery, congestive heart failure,
121]. Studies of autopsied AD brain tissue also revealed de-               cerebrovascular–carotid atherosclerosis, atrial hypotension, and
creased pyruvate dehydrogenase activity in the parietal,                   transient ischemia attacks [149]. In all these conditions, one
temporal, and frontal cortex [122–124]. Cytochrome c oxidase,              common factor is the production of oxidative stress that often
Complex IV in the electron transport chain (ETC), consistently             occurs by ischemic reperfusion injury. Patients with cardiovas-
shows decreased activity in AD brain [125]. Decreased                      cular disease and MCI are reported to have increased short-term
                                   D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677                         665


memory loss and decreased ability to learn and retain new                  elevated in MCI brain [166–168]. Our laboratory showed that
material and decreased recall and short-term memory compared               protein-bound HNE levels and 3-NT are elevated in the MCI
to MCI patients without vascular risk factors [150]. Another risk          brain compared to control IPL and hippocampus [166,169].
factor for AD is diabetes mellitus, in which increased levels of           Protein carbonyls are also elevated in MCI brain [36,167]. These
glucose and insulin may increase the levels of free radicals such          results suggest the accumulation of oxidative stress [36,108,
as superoxide and hydrogen peroxide [151]. Monomeric Aβ is                 166,167] in MCI brain and are consistent with the notion that
known to compete with insulin for insulin-degrading enzyme,                oxidative stress could be an early event in the progression of
so diabetes may lead to elevated levels of Aβ, thereby posing a            MCI to AD.
risk factor for development of AD [49]. Prevention or treatment
of vascular risk factors can reduce oxidative stress [152].                Redox proteomics identification of oxidatively modified
Vascular factors are also associated with AD [153–156].                    brain proteins in AD
Therefore, it is possible that vascular factors contribute to the
conversion of MCI to AD [157,158].                                         Redox proteomics

Oxidative stress in MCI brain                                                  Redox proteomics used in our laboratory to identify speci-
                                                                           fically oxidized proteins in Alzheimer disease brain involves
   As described above, MCI is characterized by mild current                coupling two-dimensional polyacrylamide gel electrophoresis-
memory loss without dementia or significant impairment of                  mediated separation of proteins to mass spectroscopic analysis
other cognitive functions or activities of daily living [159,160].         (Fig. 4). Two-dimensional (2D) gel electrophoresis allows the
As also noted above, a number of MCI subjects show neuro-                  analyses of complex protein mixtures based on two important
pathological hallmarks similar to AD, including temporal lobe              physicochemical properties, i.e., isoelectric focusing (IEF) and
atrophy and low CSF Aβ levels [161].                                       relative mobility [170]. The 2D gel and blot maps obtained from
   Plasma of MCI patients is reported to have decreased levels             proteomics provide information about the pI, molecular weight,
of nonenzymatic antioxidants and decreased activity of anti-               expression, and post- and cotranslational modifications of a
oxidant enzymes compared to those of controls, whereas there               protein of interest. Usually a single spot on the 2D gel
were no alterations in protein levels [162]. This diminution of            corresponds to a single protein [171]. 2D-PAGE for separation
antioxidants could possibly increase the production of free                of proteins has some important limitations, such as solubiliza-
radicals during the progression of the disease. Previous studies           tion of membrane proteins [172], highly basic proteins, and
reported increased oxidative damage in nuclear and mitochon-               inability to detect low-abundance proteins.
drial DNA in MCI, as indexed by increased levels of 8-OHdG,                    To use the 2D approach for maximum optimization,
2,6-diamino-4-hydroxy-5-formamidopyrimidine (fapygua-                      chaotropic agents, such as urea and thiourea, coupled with
nine), 8-hydroxyadenine, 4,6-diamino-5-formamidopyrimidine                 nonionic or zwitterionic detergents are used to solubilize
(fapyadenine), and 5-hydroxycytosine [163,164]. Markesbery                 proteins and avoid protein precipitation during the IEF and
and co-workers showed that subjects with MCI have higher                   the SDS gel electrophoresis. To avoid cathodic drift, immobi-
levels of isoprostanes (F2isoP) in the plasma, urine, and cere-            lized pH IEF strips are used instead of tube gels, which improve
brospinal fluid compared to those of healthy subjects [165].               protein map reproducibility between samples. Further, the use
Lipid peroxidation markers such as free HNE, protein-bound                 of narrow-range IEF strips can expand the area of interest in a
HNE, TBARS, and malondialdehyde were reported to be                        map of a proteome to give high-resolution separation.




                                             Fig. 4. Diagrammatic representation of redox proteomics.
666                                D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677


   In addition to the often-used method of 2D-PAGE, other                  analysis, but is optimal for proteins below a relatively low
separation methods, including 2D-HPLC and isotopically coded               molecular weight (b 30 kDa) [175].
affinity tags (ICAT), are also used for protein separation. The               The correct identity of the protein is determined by analysis
ICAT method is used to analyze protein expression in two                   using peptide mass data to interrogate online protein databases.
different sets of samples together by labeling the samples with            The protein sequence database SwissProt is the most commonly
different isotopes that bind to cysteine residues [173].                   used database for protein identification that is based on
   In our laboratory we used redox proteomics techniques to                computer algorithms [176] and is available gratis through the
identify oxidatively modified proteins in oxidative stress-related         Internet. Most of the various protein search databases that are
diseases and their models. In this method we use a parallel                available online are listed in Table 1.
analysis that couples 2D-PAGE with 2D-immunochemical                          The information obtained from proteomics has significant
detection of protein carbonyls derivatized by DNPH, nitrated               potential to provide insight into AD pathogenesis, to develop
proteins indexed by 3-NT, or protein adducts of HNE, on 2D                 disease markers, and to identify potential targets for drug
Western blots, followed by MS analysis of the corresponding gel            therapy in AD.
spot, as shown in Fig. 4. Proteins containing reactive carbonyl
groups/3-NT/HNE in AD and control brain samples are detected               Oxidative modification of brain proteins in AD: carbonylation
by 2D Western blot analysis using specific antibodies. The 2D              and nitration
Western blots and 2D gel images are matched by computer-
assisted image analysis, and the anti-DNP/nitrotyrosine/HNE                    Any type of oxidative modification (i.e., nitration, carbony-
immunoreactivity of individual proteins is normalized to their             lation, etc.) generally causes the protein to lose functionality and
content, obtained by measuring the intensity of colloidal                  lowers enzyme activity [12]. Oxidatively modified proteins
Coomassie blue- or SYPRO ruby-stained spots. Such analysis                 have been identified in AD hippocampus and AD IPL
allows comparison of levels of oxidatively modified brain                  [10,13,37,38,177,178]. Previous reports of an increase in total
proteins in AD versus control subjects.                                    protein carbonyls in AD brain [82] initiated studies to determine
                                                                           if specific proteins were more vulnerable or targeted for protein
Mass spectrometry and database searching                                   oxidation. Using redox proteomics, we identified specific
                                                                           carbonylated proteins in the hippocampus and IPL of the AD
    Once a protein spot is identified as significantly involved in         brain. In the IPL region, we reported dihydropyriminidase-
AD or MCI, it is digested with trypsin in the gel, and the                 related protein 2 (DRP2), α-enolase, heat shock cognate 71
resulting tryptic peptides undergo mass spectrometry analysis              (HSC 71), creatine kinase BB (CK BB), glutamine synthase
[10,12,13,36–38,174]. The peptide mass fingerprints obtained               (GS), and ubiquitin carboxy-terminal hydrolase L-1 (UCHL-1)
are characteristic of a specific protein, which allows correct             as showing a specific increase in protein carbonyls in the AD
identification of a particular protein using a suitable database           brain compared to age-matched controls [37,38]. In a separate
that compares the experimental masses with theoretical masses              study of the AD hippocampus, α-enolase, UCHL-1, DRP2,
of trypsin-generated protein sequences.                                    HSC 71, CK BB, peptidylprolyl-cis,transisomerase 1 (Pin1),
    The mass-spectrometric methods that are mostly used                    triosephosphate isomerase (TPI), and carbonic anhydrase II
include MALDI (matrix-assisted laser desorption/ionization)                (CAII) were identified as showing an increase in protein
and ESI (electrospray ionization). In MALDI analysis the                   carbonyls in the AD brain compared to age-matched control
peptide sample is mixed with a matrix (usually α-cyano-4-                  brain [12]. These data support the notion that protein carbony-
hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) and                     lation affects energy metabolism, pH regulation, and mitochon-
deposited onto a plate that is subjected to laser radiation,               drial functions.
which allows the matrix to absorb the energy and then transfer                 Protein nitration has detrimental effects as seen in key
the proton to the peptides in the gas phase. In ESI, MS/MS                 proteins such as glutamine synthase [179], ubiquitin [180],
analysis of peptides leads to sequence information of these                tyrosine hydroxylase [181,182], and Mn superoxide dismutase
tryptic peptides. The exact mechanism for the ionization process           [183–186], which lose activity upon protein nitration. As noted
in ESI is not well understood; however, a number of hypotheses             above, previous research has shown that protein nitration is
were put forward to explain the ionization process in ESI. One             associated with AD [10,11,13,90,187]. Our laboratory [10,13]
such hypothesis resulted from the use of HPLC coupled to ESI,              identified several proteins significantly nitrated in AD hippo-
which suggests that as the liquid leaves the nozzle, the electric          campus and IPL compared to control brain. These proteins
field induces a net charge on the small droplets. As the solvent
evaporates, the droplet shrinks and the charge density at the              Table 1
surface of the droplet increases. This will eventually lead to the         Mass spectrometry search engines for peptide mass fingerprinting
explosion of the droplet, producing multiply charged analyte               Search engine     URL
ions.                                                                      Mascot            http://www.matrixscience.com
    Surface-enhanced laser desorption/ionization time-of-flight            MOWSE             http://www.hgmp.mrc.ac.uk/Bioinformatics/Webapp/mowse
is another proteomics tool that couples the classical methods of           Profound          http://prowl.rockefeller.edu/profound_bin/WebProFound.exe
chromatographic sample preparation with mass spectrometry                  MS-fit            http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm
                                                                           Peptident         http://ca.expasy.org/tools/peptident.html
analysis. This method is important for applications in biomarker
                                           D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677                               667


include glyceraldehyde-3-phosphate dehydrogenase, ATP                              consequently producing NADH as a by-product of the reaction.
synthase α chain, CAII, voltage-dependent anion channel pro-                       Studies of the AD brain have shown GAPDH to be oxidatively
tein, α-enolase, γ-enolase, β-actin, L-lactate dehydrogenase                       modified with decreased enzyme activity [193,194]. LDH
(LDH), neuropolypeptide h3, and TPI (Table 2).                                     reduces pyruvate to lactate using NADH as a cofactor. A benefit
                                                                                   of this reaction is to generate NAD+, which is used as a cofactor
Functional classification of redox proteomics-identified                           for GAPDH in glycolysis. The oxidation and consequential
oxidatively modified brain proteins in AD                                          dysfunction of LDH in AD also leads to reduced GAPDH
                                                                                   activity. A reduction in GAPDH activity hinders glycolysis,
Energy-related enzymes                                                             lowering energy production in the cell. GAPDH also serves as a
    α- and γ-enolase, TPI, GAPDH, LDH, PGM1, and CK are
                                                                                       U
                                                                                   NO trap, which utilizes a large number of Cys residues [195].
seven proteins that were identified to be oxidatively modified                     Hence, oxidative modification of GAPDH would lead to a
in AD brain that affect energy production and use in the cell.                     decreased protection of neurons and increased 3-NT formation.
α- and γ-enolase are two subunits in the enolase enzyme. This                      CK BB catalyzes the phosphorylation of creatine to creatine
enzyme converts 2-phosphoglycerate to phosphoenol pyruvate                         phosphate, which is an important high-energy storage molecule
in glycolysis. α-Enolase levels are increased AD brain, but                        [196]. The phosphate group is then used in the production of
activity is reduced, causing impairment in glucose metabolism                      ATP providing immediate energy to the cell [197]. ATP is in
[12,188]. TPI catalyzes the conversion of dihydroxyacetone                         constant demand by various ATPases to maintain ion pumps,
phosphate (DHAP) to glyceraldehyde 3-phosphate (G3P) in                            lipid asymmetry, and cell–cell communication. The oxidative
glycolysis. In the previous step, fructose 1,6-bisphosphate is                     dysfunction of these enzymes identified by redox proteomics
cleaved into DHAP and G3P. Dihydroxyacetone phosphate is                           could interrupt glycolysis and disrupt energy metabolism in the
not directly involved in glycolysis. The enzyme TPI catalyzes                      brain, consistent with reduced energy utilization observed in
this reaction, converting DHAP into G3P (the aldose isomer),                       AD brain [190,191].
and causes an additional molecule of glyceraldehyde 3-phos-
phate to continue along the glycolytic pathway. TPI has been                       Proteasome-related proteins
shown to be oxidatively modified in the AD brain [12].                                 The ubiquitin–proteasome pathway has been shown to be
Another glycolytic protein, PGM1, has been shown to be                             dysfunctional in the AD brain [198]. UCHL-1 is crucial to the
oxidized in the AD brain, specifically in the hippocampus.                         degradation of damaged or misfolded proteins through pro-
PGM1 converts 3-phosphoglycerate to 2-phosphoglycerate.                            teasomal pathway. UCHL-1 has been found to be oxidized in
Glycolysis is especially important to the energy production in                     the hippocampus and IPL regions of the AD brain [12,37,199],
the brain, because glucose is the main source of energy.                           which is consistent with AD pathology [200]. The dysfunction
Impaired glycolytic function directly relates to less ATP                          of UCHL-1 would be consistent with the dysfunction of the
available to the cells and various cellular processes that require                 proteasome, accumulation of damaged and aggregated proteins,
ATP may be impaired, which is consistent with findings of                          and excess protein ubiquitinylation observed in AD [198]. Also,
altered glucose metabolism and tolerance in AD patients                            recent studies show that UCHL-1 can recover synaptic function
[189–192].                                                                         and contextual memory formation from Aβ-induced oxidation
    Glyceraldehyde-3-phosphate dehydrogenase is the enzyme                         [201]. Hence, oxidatively dysfunctional UCHL-1 may con-
responsible for converting glyceraldehyde 3-phosphate to 1,3-                      tribute to altered synaptic function and memory of AD.
bisphosphoglycerate, a high-energy phosphate product. NAD+                             The cooperation of proteasome and molecular chaperones is
and inorganic phosphate are needed to drive the reaction,                          required to control protein–protein interaction and the devel-


Table 2
Oxidatively modified proteins identified in AD hippocampus and IPL brain regions
Protein function                                  Hippocampus                      Inferior parietal lobule     Oxidative modification   Reference
Energy-related enzymes                            α-enolase ⁎, TPI, PGM1,          α-enolase, TPI ⁎, CK,        Carbonylation            [10,12,13,37,38]
                                                  GAPDH                            γ-enolase ⁎, LDH             and nitration
Proteosome-related proteins                       UCHL-1, HSC 71                   UCHL-1, HSC 71               Carbonylation            [12,37,38]
Structual proteins                                DRP2                             DRP2, β-actin ⁎              Carbonylation            [10,12,13,37]
                                                                                                                and nitration
Neurotransmitter-related proteins                                                  GS                           Carbonylation            [37]
Cholinergic function and lipid asymmetry                                           Neuropolypeptide h3 ⁎        Nitration                [10]
pH regulation protein                             CAII ⁎                                                        Carbonylation            [12,13]
                                                                                                                and nitration
Cell cycle; tau phosphorylation;                  Pin1                                                          Carbonylation            [12]
  Abeta production
Synaptic abnormalities and LTP                    γ-SNAP                                                        Carbonylation            [12]
Mitochondrial related protein                     VDAC ⁎, ATP synthase                                          Nitration                [13]
                                                  (α-chain) ⁎
 ⁎ This protein was found to be significantly nitrated.
668                               D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677


opment of aggregates [202]. The identification of the oxida-              citotoxicity. A high concentration of glutamate overactivates
tively modified HSC 71 is consistent with impairment of the               ionotropic glutamate receptors, which leads to an increase in
proteasomal pathway in AD, as HSC 71 is involved with the                 intracellular Ca2+, resulting in cell death. Synaptic depletion is
proteasome and protein degradation [38,203]. HSC 71 also                  one of the characteristics of AD brain that could be secondary to
directly binds to the cytoplasmic domain of APP, which                    glutamate excitotoxicity. Glutamine synthetase is an enzyme
conceivably could contribute to alterations in APP processing             identified by redox proteomics as being oxidatively modified in
and Aβ production observed in the AD brain [204].                         AD brain [37]. GS catalyzes the reaction of glutamate with
                                                                          ammonia to form glutamine, a nontoxic amino acid. Previous
Structural proteins: β-actin and DRP-2                                    studies have shown GS to be oxidatively modified in the AD
   Maintaining cytoskeletal integrity is important, because               brain [37]. Further studies have also revealed reduced GS
shortened dendritic length can be associated with AD pathology            activity in the AD brain [6,82]. These results are consistent with
[205]. Actin is a major component of thin microfilaments. β-              those of others, who showed that excessive extracellular
Actin plays a role in preserving cytoskeletal structure. Because          glutamate leads to intracellular ROS [219]. Decreased GS
β-actin is carbonylated [206] and nitrated in AD brain [10],              activity and excitotoxicity lead to neurodegeneration, consistent
structural integrity could be compromised and these changes               with synaptic loss in AD.
may contribute to neuronal death [22].
   DRP2 is a neuronal repair protein identified as oxidized in            Synaptic abnormalities and long-term potentiation
AD brain [12,38]. DRP2 modulates collapsin activity, which is                Soluble N-ethylmaleimide-sensitive factor (NSF) attach-
essential to elongating and directing dendrites to adjacent               ment proteins are highly conserved and are involved in
neurons. Collapsin is also critical in axonal outgrowth.                  intracellular membrane fusion and vesicular trafficking, which
Oxidative modification of DRP2 may to lead to the observed                are critical to the function of the synapse. These proteins are
shortened dendrite length in AD [207], with consequent                    involved in neurotransmitter release, hormone secretion,
impairment of neuronal communication.                                     mitochondrial organization, and vesicular transport. γ-Soluble
   DRP2 is downregulated in Down syndrome and AD patients                 NSF attachment protein is one of three isoforms and is
[208]. Shortened dendritic length observed in AD [207]                    involved in long-term potentiation, a key process for learning
conceivably could be a result of loss of function of oxidatively          and memory [220]. γ-SNAP was identified as oxidatively
modified DRP2. The protein DRP2 was also identified as                    modified in the AD brain [12], which has clear implications in
oxidized in the inferior parietal lobule of AD patients and in            AD pathology through impaired neurotransmission [221]. This
Aβ-treated synaptosomes [38,209]. Neuronal miscommunica-                  finding also has implications with respect to long-term
tion is a likely contributory factor in the memory loss and               potentiation learning and memory, the diminution of which
cognitive decline seen in AD patients as demonstrated in those            is a hallmark of AD.
with the ApoE4 allele, a risk factor for AD [210]. These redox
proteomics results are consistent with the memory and synapse             pH regulation protein
loss attributed to AD as a consequence of the decreased function              Carbonic anhydrase II catalyzes the hydration of CO2 to
of these two structural proteins.                                         HCO3−, regulates pH in the cell, and aids in transporting HCO3−
                                                                          and CO2. The main function of CAII is maintaining electrolyte
Cholinergic function and lipid asymmetry                                  and water balance [222,223]. The activity of CAII is diminished
    Neuropolypeptide h3, also known as RAF kinase inhibitor,              in AD hippocampus [178,188]. Nitration of CAII can dereg-
phosphatidylethanolamine-binding protein (PEBP), and hippo-               ulate pH in the cell. Because so many reactions and enzymes, as
campal cholinergic neurostimulating protein (HCNP), is critical           well as the mitochondrial proton gradient for ATP synthesis, are
to the production of choline acetyltransferase, which is vital            pH-dependent, malfunction of this enzyme, carbonic anhydrase
in signal transduction. The loss of choline acetyltransferase             II, may be involved in AD.
leads to reduced levels of the neurotransmitter acetylcholine,
causing poor neurotransmission [211]. More specifically, there            Cell cycle, tau phosphorylation, Aβ production
is decreased expression of HCNP in AD hippocampus [212].                     Pin1 is one of the peptidylprolyl isomerases that regulate
N-methyl-D-aspartic acid (NMDA) receptors activate the                    biological functions of proteins, including protein assembly,
production of this enzyme, and alteration of the NMDA receptor            folding, intracellular transport, intracellular signaling, transcrip-
mediates cholinergic deficits [213]. Alzheimer disease is                 tion, cell cycle progression, and apoptosis. Of particular
associated with cholinergic neuronal loss [214–216]. Because              relevance to AD, Pin1 regulates enzymes associated with
neuropolypeptide h3 is also PEBP, it is possible that lipid               both phosphorylation and dephosphorylation of the critical
asymmetry is also affected by oxidized PEBP. We showed that               cytoskeletal protein, tau [224]. Also, Pin1 binding to APP
lipid asymmetry is lost in synaptic membranes treated with                regulates the production of Aβ [225]. Finally, Pin1 is related to
Aβ(1–42) or HNE [217,218].                                                neurons in the cell cycle [224]. Because, as we have shown, Pin1
                                                                          is oxidatively dysfunctional in both MCI and AD brain
Neurotransmitter-related proteins                                         [12,36,177], three major pathological hall marks of this
   An excess of the excitatory neurotransmitter glutamate                 dementing disorder (NFT, SPs, and synapse loss due to neurons
causes toxicity to neurons by overstimulating them, e.g., ex-             trapped in the cell cycle) may be related to damaged Pin1. In
                                   D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677                           669


MCI brain, elevation of cell cycle proteins was found [226],               Redox proteomics analysis of in vivo models of AD centered
consistent with this notion. In addition, previous studies reported        around human Aβ(1–42)
downregulation of Pin1 levels and activity in AD brain [36,178].
Pin1 was reported to colocalize with phosphorylated tau in AD              Human Aβ(1–42) injected into rat cholinergic-rich basal
brain and showed an inverse relationship to the levels of tau. In          forebrain
addition, an in vitro study reported that Pin1 protects neurons
against age-related neurodegeneration [227]. Pin1 can also                     A cholinergic animal model of AD was prepared by injecting
restore the ability of phosphorylated tau to bind microtubules             Aβ(1–42) into the nucleus basalis magnocellarius (NBM) of rat
and promote their assembly in vitro, a process that might                  brain to replicate the cholinergic dysfunction reported in AD
represent a potential therapeutic use for Pin1 [228]. Studies to           brain [235,236]. Degeneration of the basal forebrain cholinergic
test the hypothesis of the critical importance of Pin1 in AD               neurons is pronounced in AD and is associated with cognitive
pathogenesis are ongoing in our laboratory.                                deficits [237,238]. In this animal model oxidative stress was
                                                                           observed in hippocampus, cortex, and nucleus basalis; however,
Mitochondrial-related proteins                                             an extensive protein oxidation was observed in hippocampus
   Voltage-dependent anion channel protein is the outer                    compared to other regions [35,237].
component of the mitochondrial permeability transition pore                    Using regional redox proteomics, a large number of oxidized
(MPTP). VDAC is critical to importing and exporting various                proteins were identified in this animal model that include 14-3-
metabolites (i.e., ATP) into the mitochondria. This protein, in its        3ζ, β-synuclein, pyruvate dehydrogenase, GAPDH, PGM1,
role as a part of the MPTP, plays a part in the release of                 GS, and tubulin (β-chain 15/α) and chaperonin 60 (HSP60)
cytochrome c [229], caspases [230], and apoptosis-inducing                 [35]. These proteins play a crucial role in signal transduction,
factor [231] from the mitochondria, essential for apoptosis.               cellular structure, energy metabolism, and stress response. As
Nitration of VDAC alters MPTP function and can induce                      discussed above, a number of proteins that were identified in
apoptosis by preventing Bcl-2/VDAC interaction in favor of                 this animal model were already reported to be oxidatively
increasing the proapoptotic factor Bax and cytochrome c release            modified in AD brain, including PGM1, GAPDH, GS, and
[232]. Thus neuronal death could ensue. Recent research                    tubulin [10,12,13]. The expression of 14-3-3 proteins was
suggests that VDAC plays a role in Aβ-mediated neurotoxicity               reported to be increased in AD brain and CSF [239,240] and
[233].                                                                     these proteins also were found to be associated with NFTs
   ATP synthase, α subunit, is an ATP-synthesizing enzyme                  [241].
composed of a coupling factor 1 (F1) head and the hydrophobic                  Recently, it has been shown that 14-3-3ζ acts as a scaffolding
F0 component. The F1 head is composed of three catalytic sites             protein, simultaneously binding to tau and glycogen synthase
for ATP production. Once ADP and inorganic phosphate (Pi)                  kinase 3β in a multiprotein tau phosphorylation complex [242].
bind to the catalytic site of this enzyme, one ATP is created. This        The hyperphosphorylation of tau could be associated with
binding of ADP to Pi must be in a precise “tight” conformation             the oxidation of 14-3-3ζ in this rat model after Aβ(1–42)
for ATP to be produced. If the ADP and Pi are in the “loose” or            injection into NBM, which could unite both the importance
“open” conformation, their affinity is not high enough for ATP             of Aβ(1–42) and the hyperphosphorylation of tau. Heat
production. Therefore, ATP production through ATP synthase                 shock protein 60 is a mitochondrial chaperone protein that is
occurs in a “rotary catalysis and proton electrochemical                   involved in mediating the proper folding and assembly of
gradient” [234]. The rotation occurs by protons inducing a                 mitochondrial proteins, especially in response to oxidative
conformational shift. Loss of function of this protein can be              stress [243]. Expression of HSP60 is significantly decreased
detrimental to ATP production and in energy metabolism,                    in AD, and Aβ(25–35) has been shown to induce oxidation
consistent with PET studies of AD.                                         of HSP60 in fibroblasts derived from AD patients compared
                                                                           to age-matched controls [203,244]. The proteomics identifi-
Redox proteomics: identification of oxidatively modified                   cation of the oxidation of HSP60 by Aβ(1–42) in vivo [35]
brain proteins in MCI                                                      could presumably lead to loss of function of HSP60 that may
                                                                           lead to increased protein misfolding and aggregation, as well
    A recent redox proteomics study from our laboratory                    as an increased vulnerability to oxidative stress. Moreover,
identified α-enolase, glutamine synthetase, pyruvate kinase                as noted above, in AD there is evidence that Aβ(1–42)
M2, and Pin1 in MCI hippocampus as being oxidatively                       migrates to mitochondria, where HSP60 conceivably could
modified [36]. In AD brain, three of these proteins, i.e., Pin1,           be affected.
glutamine synthetase, and enolase, were reported as oxidatively
modified [37,38,177]. These proteins are crucial for energy                Human Aβ(1–42)-expressing Caenorhabditis elegans
metabolism and neurotransmission. The identification of Pin1
as a common target of oxidation between AD and MCI suggests                   Transgenic C. elegans is a worm model in which human
that Pin1 oxidation might be one of the driving forces for the             Aβ(1–42) is expressed through a body-wall muscle myosin
initiation or progression of AD pathogenesis that may                      promoter and an Aβ minigene [245]. This model has been used
ultimately lead to cell death and neurodegeneration. Research              to study Aβ toxicity, fibril formation, and oxidative stress in
to test this notion is ongoing in our laboratory.                          vivo [50,246,247]. Increased protein oxidation that preceded
670                               D.A. Butterfield et al. / Free Radical Biology & Medicine 43 (2007) 658–677


fibrillar deposition of human Aβ(1–42) was observed,                      GST were found to be commonly oxidized between AD and
consistent with the notion that small, soluble oligomers of the           aged beagle dogs.
peptide are the toxic species [50]. We also showed that C.                   The in vivo models of Aβ support a critical role for Aβ-
elegans expressing human Aβ(1–42) exhibited an increased                  induced oxidative stress and consequently the role of Aβ in AD
oxidative stress and also suggested association of methionine 35          pathology. The beagle studies suggest a possible therapeutic
of Aβ(1–42) in the mechanism of oxidative damage [247]. A                 approach to lowering the risk of developing AD that combines
redox proteomics study in this animal model identified 16                 behavioral enrichment with an antioxidant-rich diet that, similar
proteins that were oxidatively modified [34]. The oxidatively             to the aged beagles with Aβ(1–42) of the same sequence as
modified proteins in this model of Aβ include ATP synthase α              humans, could delay or significantly modulate AD pathology
chain, glutamate dehydrogenase, proteasome α and β subunit,               while improving memory.
glutathione S-transferase (ε class), medium- and short-chain
acyl-CoA dehydrogenase, arginine kinase, myosin regulatory                Future research
light chain, actin, adenosine kinase, malate dehydrogenase,
transketolase, translation elongation factor EF-1γ, lipid-binding            The increased oxidative stress parameters in MCI brain
protein, and RACK1 ortholog. These identified proteins were               regions [36,166,167,169] suggest that oxidative stress may be
involved in a variety of cellular functions including energy              an early event in the progression from normal brain to AD
metabolism, protein degradation, cytoskeletal integrity, antiox-          pathology. These results further support the hypothesis that
idant system, signal transduction, and lipid metabolism. Many             oxidative stress is a mediator of synaptic loss and a presumed
of these classes of oxidized proteins are also represented in AD          factor for the formation of neurofibrillary tangles and senile
brain as noted above.                                                     plaques [10,12,13,37,38,98,199]. A better understanding of
                                                                          MCI could help in delineating the mechanism of AD and in
Canine model                                                              developing effective therapeutics to slow or prevent conversion
                                                                          of MCI to AD and to treat AD. Recent AD clinical research has
   Beagle dogs are a good model to study the role of Aβ,                  emphasized early detection of AD with the hope that early
because these animals have the same Aβ sequence as humans.                treatment will slow or prevent the progression of AD
In addition, aged beagles deposit Aβ and have cognitive                   pathogenesis. Ongoing efforts in our laboratory include testing
dysfunctions similar to humans [248,249]. Histopathologically,            of promising therapeutic approaches to inhibit oxidative
aged beagle canines show reduced cerebral volume, cortical                damage in MCI, AD, and models thereof [29,60,261–268].
atrophy, and ventricular widening as assessed by in vivo                  Given the expected 14–16 million Americans and more than
magnetic resonance imaging [250–252]. Beagle dogs absorb                  22 million persons worldwide expected to develop AD in
dietary nutrients in ways similar to those of humans. Previous            the relatively near future in the absence of effective inter-
studies in aged canines showed that long-term antioxidants                vention, increased national funding for AD-related research is
reduce cognitive decline [253–255] and lead to rapid improve-             imperative.
ments in complex learning ability in aging dogs [256,257].
These characteristics make the beagle dog highly relevant for             Acknowledgments
studying human AD considering the similarities in Aβ
deposition, histopathology, and nutrition. The histopathological             This work was supported in part by grants from the National
results in aged beagle dog imitate human AD brain pathology               Institutes of Health (AG-05119, AG-10836).
before any occurrence of amyloid deposition [258–260].
   Using redox proteomics, our group identified six brain                 References
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