Brain Protein Oxidation in Alzheimer’s Disease 595
was recently confirmed by others (Choi et al., 2004). Taken together, these differ-
ent lines of evidence support a role for dysfunction of the ubiquitin–proteasome
pathway in the pathogenesis of AD. Others showed that diminished proteasome
function could l ead to neurodegeneration (Halliwell, 2002) and oxidative stress
(Ding et al., 2003). On the other hand, oxidative stress leads to proteasome dysfun-
tion (Halliwell, 2002, 2006), suggesting a vicious feedforward cycle of oxidative
stress, proteasome dysfunction, and neurodegeneration.
Neuropolypeptide h3 (NPH3), a phosphatidyloethanolamine-binding protein
[PEBP] or cholinergic neurostimulating peptide, may play an important role in reg-
ulating choline acetyl transferase (ChAT) and maintaining phospholipid asymmetry,
a process that is important to normal mitochondrial and plasma membrane function
(Castegna et al., 2004a; Mohmmad Abdul and Butterfield, 2005). Oxidation of this
protein could lead to impaired cholinergic properties, mitochondria function, and
apoptosis in AD.
β-actin (ACT) and dihydropyrimidinase-related protein 2 (DRP2) were found to
be downregulated and oxidatively modified in AD brain (Coleman and Flood, 1987;
Lubec et al., 1999; Castegna et al., 2002a,b, 2003). Alterations in the structure of
proteins induced due to oxidation could be one of the contributing factors involved
in the observed loss of interneuronal connections, neuronal repair, and shortened
dendritic lengths in AD brain (Coleman and Flood, 1987), conceivably leading to
memory impairment and synapse loss, clearly important for AD.
Another important protein that is found to have reduced expression and is also
oxidized and has reduced activity in AD brain is peptidyl-prolyl cis/trans isomerase
(Pin1). This protein is colocalized with phosphorylated tau (Holzer et al., 2002;Kurt
et al., 2003; Ramakrishnan et al., 2003; Sultana et al., 2006c, d). Pin1 is a chaperone
enzyme that recognizes phosphorylated Ser-Pro and phosphorylated Thr-Pro motifs
in proteins, and alters the conformation of proteins from cis to trans between a given
amino acid and a proline (Schutkowski et al., 1998). One of the target proteins of
Pin1 is a protein that removes phosphate moieties from tau (Shen et al., 1998).
Oxidative modification of Pin1 may lead to hyperphosphorylation of tau, and entry

into a cell cycle eventually leading to tangle formation and apoptosis (Nagy et al.,
1997; Zhou et al., 2000; Smith et al., 2004). In addition to a role of Pin1 in neu-
rofibrillary tangle formation, recent studies suggest that Pin1 plays a role in APP
processing, and therefore, in Aβ levels in brain (Pastorino et al., 2006). Thus, Pin1
is involved in two of the major pathological hallmarks of AD. Pin1 is oxidatively
modified and dysfunctional in mild cognitive impairment (MCI), a precursor condi-
tion to AD (Butterfield, 2006). Further studies are required to understand the role of
Pin1 in the disease progression.
Soluble N-ethylmaleimidesensitive factor (NSF) attachment protein (γ-SNAP)
is another protein found to be oxidatively modified in AD brain, and this protein is
important in vesicular transport for neurotransmitter release, hormone secretion, and
mitochondrial integrity. Hence, oxidation may lead to an altered neurotransmission
system and impaired learning and memory in AD (Masliah et al., 1994; Scheff and
Price, 2003; Sultana et al., 2006d).
596 R. Sultana and D.A. Butterfield
The pH of the cell is crucial for the normal functioning of the cells. Carbonic
anhydrase 2 (CA2) regulates cellular pH, CO
2
, and HCO
3
–
transport, and maintains
H
2
O and electrolyte balance (Sly and Hu, 1995) by reversible hydration of CO
2
in
normal cells. This protein has been reported to be oxidized in AD brain and also
showed a decrease in activity (Meier-Ruge et al., 1984; Poon et al., 2004; Sultana
et al., 2006d). Functionally inactive CA2 could induce changes in buffering systems

in the brain, which could consequently lead to protein aggregation. Protein aggre-
gation is more pronounced in AD brain, and, because cellular pH could be altered,
altered mitochondrial production of ATP could be affected.
The voltage-dependent anion channel (VDAC) is identified as one of the oxi-
dized proteins in AD brain (Sultana et al., 2006a,b). The oxidation of this protein
in AD suggests an alteration in the function of the mitochondrial permeability
transition pore (MPTP) leading to mitochondrial depolarization and altered sig-
nal transduction pathways, which could be crucial in synaptic transmission and
plasticity. Moreover, alterations in the MPTP could lead to apoptotic processes.
In addition, dysfunction of mitochondria recently has been reported to alter APP
metabolism, enhancing the intraneuronal accumulation of amyloid β-peptide and
enhancing neuronal vulnerability (Busciglio et al., 2002).
Overall, from the data presented above it is clear that oxidation of specific brain
proteins alters the structure and thereby function of the proteins. Such changes could
be important in AD pathology.
4 Is Protein Oxidation an Early or Late Event
in AD Pathogenesis?
In recent years, the clinical stages preceding AD presenting memory impairment
but without overt dementia have attained increased attention in the AD clinical and
research fields. Patients with MCI are subjects with memory or other cognitive com-
plaints but who do not fulfill the dementia criteria (Visser et al., 2001). Persons with
MCI represent a heterogeneous group of patients with several possible explanations
for the cognitive deficits. A high proportion of MCI patients are probably early
AD subjects, although other diagnoses are also included in this diagnostic entity.
Biochemical markers for AD should reflect the pathogenesis of the disorder.
Both in MCI and AD patients, mean plasma levels of nonenzymatic antioxi-
dants and activity of antioxidant enzymes appeared to be lower than in controls,
with no parallel induction of antioxidant enzymes (Keller et al., 2005). In order to
explain these results it has been suggested that the increased free radical production
in MCI might lead to a rapid consumption of plasma antioxidants without a simul-

taneous activation of new molecules of antioxidant enzymes. Individuals with MCI,
and subsequently with AD, are likely to have an inadequate antioxidant enzymatic
activity, unable to counteract the increased production of free radicals during the
pathogenesis of the disease.
Brain Protein Oxidation in Alzheimer’s Disease 597
Subjects with MCI have increased protein oxidation in hippocampus and IPL
(Butterfield et al., 2006a) and superior and medial temporal gyri (Keller et al.,
2005). Additionally, using redox proteomics we identified three specific proteins,
that is, enolase, glutamine synthase, and Pin1 as common targets of protein oxida-
tion between MCI and AD which suggests that protein oxidation of these selected
proteins could be important in initial events involved in AD pathogenesis (Fig. 5).
Furthermore, several gene mutations associated with AD have been observed in
subjects with MCI including mutations in apolipoprotein E, presenilin 1, and the
amyloid precursor protein (Traykov et al., 2002; Nacmias et al., 2004). Increased
levels of lipid peroxidation have been reported in the brain of persons with MCI
(Keller et al., 2005; Markesbery et al., 2005; Butterfield et al., 2006b). Thus,
increased levels of protein and lipid peroxidation could be implicated as early events
in AD pathophysiology and also suggest that pharmacological intervention to pre-
vent protein and lipid peroxidation at the MCI stage or earlier may be a promising
therapeutic strategy to delay or prevent progression to AD.
AD
gamma-Enolase
alpha-enolase
glutamine
Creatinine Kinase
Triose Phosphate Isomerase
Phosphoglycerate mutase 1
Ubiquitin carboxy-terminal
hydrolase L-1
Heat Shock Cognate 71

Dihydropyrimidinase-related
protein 2
Gamma-SNAP,
Carbonic anhydrase 2
MCI
synthetase
peptidyl-prolyl
cis/trans isomerase 1.
Pyruvate kinase M2
Fig. 5 Comparison between the MCI and AD brain to see the common targets of protein oxidation
Very recent studies reported increased oxidative damage in nuclear and mito-
condrial DNA in MCI, as indexed by increased levels of 8-hydroxyguanosine
(8-OHdG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (fapyguanine), 8-hyd-
roadenine, 4,6-diamino-5-formamidopyrimidine (fapyadenine), and 5-hydroxy-
cytosine (Wang et al., 2006). Due to the crucial role that DNA plays in cells,
high levels of oxidation, particularly early in the progression of AD, may result
in a decline of normal cell function through altered transcription, changes in pro-
tein expression, or cross-linking with proteins. Taken together, these results suggest
that oxidative damage is one of the factors involved in the pathogenesis of neu-
rodegeneration in AD and is not simply a late effect of the neurodegenerative
process.
598 R. Sultana and D.A. Butterfield
5 Conclusions
Protein oxidation may be an early event in AD pathogenesis as supported by the
data from the MCI brain. With exceptions related to oxidative signaling or other
beneficial processes, excessive oxidation of proteins has been reported to decrease
the functionality of most proteins, which suggests that protein oxidation is harmful
for cell survival. Further studies are in progress to understand the role of protein
oxidation and its abrogation in AD by using in vivo and in vitro models of AD
centered around Aβ(1-42) (Sultana et al., 2006a).

Acknowledgments This work was supported in part by NIH grants [AG-10836; AG-05119].
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