10.1. MILD COGNITIVE IMPAIRMENT

Mild cognitive impairment (MCI) is generally referred to as the transitional zone between normal cognitive aging and early dementia or clinically probable Alzheimer’s disease (AD) (1), although not all AD patients pass through an MCI stage. The term was first coined by Petersen (2). Most individuals with MCI eventually develop AD, which suggests MCI may be the earliest phase of the AD (3–6). MCI can be divided into two broad subtypes: amnestic (memory-affecting) MCI or non-amnestic MCI (2,7). Other functions, such as language, attention, and visuospatial skills, may be impaired in either type. Amnestic mild MCI patients characteristically have subtle but measurable memory disorder not associated with dementia. Individuals with MCI are at an increased risk of developing AD, or another form of dementia with a rate of progression between 10% and 15% per year (8,9), although there have been cases where patients have reverted to normal (1,10,11).

Criteria for MCI include (a) a memory complaint corroborated by an informant; (b) objective memory test impairment (age and education adjusted); (c) general normal global intellectual function; (d) activities of daily living not disturbed; (e) clinical dementia rating (CDR) score of 0.0 to 0.5; (f) no dementia; and (g) a clinical evaluation that revealed no other cause for memory decline (12). Moreover, neuroim-aging studies by magnetic resonance imaging (MRI) demonstrate the atrophy of the hippocampus or entorhinal cortex in MCI patients, indicating the relationships with transition of normal aging to MCI, then later to clinical AD (13). Pathologically, MCI brain shows mild degradation of the hippocampus, entorhinal cortex, sulci, and gyri using MRI (14,15). These aforementioned areas undergo considerable degradation in AD (16–20). Since the hippocampus is the region of the brain primarily responsible for processing memory, it is clearly understandable why those persons with AD and MCI have memory loss.

10.2. OXIDATIVE STRESS

Under normal physiological conditions, there is equilibrium between the level of antioxidants and pro-oxidants in a cell. However, when environmental factors, stressors, or disease occur, this homeostasis can become imbalanced in favor of pro-oxidants, resulting in a phenomenon known as oxidative stress (21). Oxidative stress can also transpire through an antioxidant deficiency (22) or excess reactive oxygen species/ reactive nitrogen species (ROS/RNS) production. Moreover, oxidatively damaged proteins are often removed by the 20S proteosome. Defects in the proteosome system would lead to elevated levels of oxidatively modified proteins and neurotoxicity (21). Oxidative stress plays a significant role in neurodegenerative disease (23–27). If oxidative stress is involved in the progression of AD and is not a consequence of this disease, there should be evidence of elevated oxidative stress in the beginning stages of the disease (28). Manifested by elevated levels of nucleic acid oxidation, protein oxidation, and lipid peroxidation, oxidative damage is most severe in the hippocampus, a brain region that is responsible for memory processing and cognitive function (23,24). Moreover, oxidative stress-mediating entities per se induce neuronal death in vitro, and protein oxidation and lipid peroxidation in the superior and middle temporal gyri (SMTG) of MCI patients are increased (29). All of these studies strongly suggest that oxidative stress is a primary event in the development of AD.

10.3. POST-TRANSLATIONAL MODIFICATIONS

Post-translational modifications (PMTs), such as protein oxidation, are seen in a plethora of neurodegenerative diseases (23,30). A few examples of PMTs are phosphorylation, nitration, carbonylation, 4-hydroxy-2-nonenal (HNE) modification, gly-cosylation, and S-glutathionylation. Proteins are sensitive to oxidation by ROS and RNS, and oxidative damage is a result from such interaction. Protein oxidation leads to loss of protein function and often cell death via necrotic or apoptotic processes (30). Protein oxidation is increased in MCI (31,32) and will be discussed thoroughly in the next section. A complete list of oxidatively modified proteins in MCI brain determined by redox proteomics can be seen in Table 10.1.

TABLE 10.1

Proteins Oxidatively Modified in MCI.

10.4. PROTEIN CARBONYLATION

Protein carbonyl levels have been recognized as the most widely used indicator of oxidative stress (33–35). Protein carbonyl levels increase in aging and age-associated neurodegenerative diseases (36,37). Proteins can be oxidized directly by reactive oxygen species to generate protein carbonyls. This happens by several different mechanisms including amino acid side chain (Lys, Thr, Pro, and Arg) and metal-assisted oxidation (38) (Figure 10.1). Beta scission from the peptide backbone is the second method by which protein carbonyls are created (Figure 10.2). Third, the adduction of carbonyl-containing reactive aldehydes (i.e., acrolein, 4-hydroxynonenal) to amino acids leads to carbonyls (Figure 10.3). Advanced glycation end products resulting from Amadori chemistry can also lead to protein oxidation (39,40). Indexing protein carbonyl content is a fundamental key to understanding protein oxidation. Protein carbonyl levels most often are analyzed by immunochemical detection of the hydrazone formed by reaction of 2,4-dinitrophenylhydrazine with protein carbonyls (30,38).

FIGURE 10.1

Beta scission from peptide backbone.

FIGURE 10.2

Amino acid side chain oxidation.

FIGURE 10.3

Covalent modification of amino acids by Michael addition.

10.5. HNE MODIFICATION

Lipid peroxidation is a complex process involving the interaction of oxygen-derived free radicals with polyunsaturated fatty acids, resulting in a variety of highly reactive electrophilic aldehydes that are capable of easily attaching covalently to proteins by forming adducts with cysteine, lysine, or histidine residues (41) through Michael addition (30). Among the aldehydes formed, malondialdehyde (MDA) and HNE represent the major products of lipid peroxidation (41). The brain is particularly vulnerable to lipid peroxidation (42) due to its richness in polyunsaturated fatty acids, high oxygen consumption, and abundant quantities of redox transition metals (43,44).

Lipid peroxidation is highly evident in neurodegenerative disease (24,45,46). Lipid peroxidation occurs through ongoing free radical chain reactions until termination occurs (Figure 10.4). Free radicals attack an allylic hydrogen atom to form a carbon centered radical (step 1). This radical reacts with O₂ to produce peroxyl radicals (step 2). These peroxyl radicals can react with adjacent lipids forming a lipid hydroperoxide repeating the cycle (step 3). The lipid hydroperoxide can decompose to produce multiple reactive products such as acrolein, malondialdehyde, and HNE. In MCI, acrolein and HNE have been found to be significantly elevated (31,47,48). Lipid peroxidation can be terminated by two radicals reacting forming a nonradical and oxygen (step 4). HNE is a major product of lipid peroxidation and causes cell toxicity. Lipids are particularly vulnerable to oxidation due to the fact that polyunsaturated fatty acids are abundant in brain and the presence of oxygen in the lipid bilayer is at millimolar levels. Glutathione has been shown to detoxify HNE in cells (49). Increased levels of HNE cause disruption of Ca²⁺ homeostasis, membrane damage, and cell death (41). Vitamin E (alpha-tocopherol) is a “chain breaking” antioxidant and can terminate propagation steps of lipid peroxidation. When the hydrogen is abstracted in step 1, an alpha-tocopherol radical forms that can be reverted back to vitamin E by ascorbic acid (vitamin C) or glutathione (GSH), both potent antioxidants.

FIGURE 10.4

Lipid peroxidation reaction summary.

HNE is an alpha, beta-unsaturated alkenal product of omega-6 polyunsaturated fatty acids and is a major cytotoxic end product of lipid peroxidation that mediates oxidative stress-induced death in many cell types (41,50,51). HNE accumulates in membranes at concentrations of 10 μM to 5 mM in response to oxidative insults (41) and invokes a wide range of biological activities, including inhibition of protein and DNA synthesis (52–56), disruption of Ca²⁺ homeostasis, membrane damage, cell death (41), and activation of stress signaling pathways (50,57).

Several publications report that the brains of MCI patients are under oxidative stress. Increased levels of thiobarbituric acid reactive substance (TBARS), malon-dialdehyde (MDA), F₂ isoprostanes and F₄ neuroprostanes, as well as soluble and protein-bound HNE, specific markers of in vivo lipid peroxidation (30), were significantly elevated in cerebrospinal fluid (CSF), plasma, urine, and brains of MCI patients compared with controls (29,31,46,58), suggesting that lipid peroxidation may be an early event in the pathogenesis of the disease.

10.6. PROTEIN NITRATION

In conjunction with the enzyme nitric oxide synthase, arginine is converted into nitric oxide and L-citrulline (Figure 10.5). Nitric oxide can react with superoxide radical anion (O₂-) forming the strong oxidant peroxynitrite (Figure 10.6). Peroxynitrite can oxidize tyrosine, methionine, tryptophan, and cysteine residues to promote protein nitration (59,60). In the presence of CO₂^, peroxynitrite can exist as an anion (ONOO) or other reactive intermediates. A nitrosoperoxyl intermediate is formed from the combination of peroxynitrite and carbon dioxide, which rearranges to form nitrocarbonate. This species can be cleaved to form carbonate and NO₂ radicals (Figure 10.6); the latter reacts with a tyrosyl free radical in the 3-position to form 3-nitrotyrosine (Figure 10.7).

FIGURE 10.5

Production of nitric oxide from L-citrulline.

FIGURE 10.6

Combination reaction of peroxynitrate and carbon dioxide.

FIGURE 10.7

Formation of 3-nitrotyrosine.

Previous research has shown that peroxynitrite can interact with proteins (61,62), lipids (63), DNA (64,65), and RNA (66) to promote damage in these biological molecules. Tyrosine nitration is associated with Alzheimer’s disease (AD) (67) as well as Parkinson’s disease (68). Gamma-glutamylcysteinylethyl ester (GCEE), a derivative of gamma-glutamylcysteine, can cross the blood-brain barrier (BBB) and has been proven to prevent peroxynitrite-induced damage by upregulating glutathione production (69). Lipoic acid has also been used to reduce protein nitration (70) as has gamma-tocopherol (71). Increased protein nitration also can lead to an elevated release of RNS. Nitration of proteins results in the inactivation of several important mammalian proteins such as MnSOD (72–75), glyceraldehyde 3-phosphate dehydrogenase (76,77), actin (78,79), synaptic proteins (80), and tyrosine hydroxylase (81,82), among others.

10.7. OXIDATIVE DAMAGE IN MCI BRAIN

Protein oxidation has been observed in brains of patients with MCI (32). Increased protein-bound HNE immunoreactivity is observed in the MCI hippocampus and MCI inferior parietal lobule (IPL) (31) as well as elevated levels of protein carbonyls (83) and 3-nitrotyrosine (32). Since MCI is arguably the earliest form of AD and there is supporting evidence of oxidative damage in the AD brain, these data suggest that elevated levels of oxidative stress in MCI can contribute to the progression of this neurodegenerative disease.

10.8. REDOX PROTEOMICS

The proteome is the complete set of proteins expressed in a cell or organism. This term was first coined by Wilkins et al. in 1996 (84). Proteomics is the analysis of the proteome. Proteomics describes a protein in terms of its amino acid sequence, mass, isoelectric point (pI), and structure. This technique is helpful in determining protein identification, modification, and function. Since the advent of proteomics, this methodology is one of the fastest growing areas of biomedical science. Proteomics is widely used to determine potential biomarkers in neurodegenerative diseases, specifically AD (85–87). Technology in the field of proteomics is rapidly expanding and evolving. Proteomics is now being developed for clinical diagnosis (88,89) and in establishing biomarkers for disease including MCI (87,90,91) and AD (86,92).

10.8.6. Database Searching

Better known as peptide mass fingerprinting (PMF), this process requires the use of MS analysis to determine the experimental masses. Experimental masses are used by on-line protein databases to compare and match protein-specific mass fingerprints generated by the in silico digestion of proteins. This comparison is used to identify the protein of interest based on the quality of peptide matches (97–99). PMF is used to identify proteins from tryptic peptide fragments by utilizing an appropriate search engine (Table 10.2, and Chapter 5). Database searches are conducted allowing for up to one missed trypsin cleavage and using the assumption that the peptides are monoisotopic, oxidized at methionione residues, and carbamiodomethylated at cysteine residues. Mass tolerance of 150 ppm/g is the window of error allowed for matching the peptide mass values. Probability-based MOWSE scores are estimated by comparison of search results against estimated random match population and are reported as -10 * Log₁₀ (p), where p is the probability that the identification of the protein is incorrect. As a correlate, the higher the MOWSE score, the lower the p value, yielding a higher probability of accurate protein identification. All protein identifications must be reviewed (and in most cases validated; for this see Chapters 11 and 14) to ensure that they are in the expected size and pI range based on position in the gel.

TABLE 10.2

Listing of Online Search Engines.

10.9. ENZYME ASSAYS

Enzymatic activity is generally lower in oxidatively modified enzymes compared to control (31,100,101). Oxidative stress leads to oxidative modification of key proteins and enzymes (102). Oxidative modification of amino acids, particularly at the active site, can cause loss of enzymatic activity and subsequent protein dysfunction (100,103–105). Therefore, the determination of enzyme activity is frequently used to help validate the identities of the proteomics-identified proteins.

10.10. ENERGY-RELATED PROTEINS

Glycolysis is a metabolic pathway that converts glucose to pyruvate and generates two ATP molecules in the process. ATP, the energy source of the cell, is extremely important at nerve terminals for normal neural communication. Decreased levels of cellular ATP at nerve terminals may lead to loss of synapses and synaptic function, and may ultimately contribute to memory loss in amnestic MCI patients. Synapse loss is an early event in AD, since it is observed in MCI and early AD (106). Energy metabolism alteration is a current major hypothesis of AD and has been reported in brain in an advanced stage of MCI (107–110). Several proteins identified by proteomics as energy-related proteins in MCI brain support this notion. There is an assortment of energy-related proteins that have been identified as being oxidatively modified in MCI hippocampus and IPL in addition to previous proteomics studies of AD and cell culture models of AD, identifying an increased oxidation of enolase (111–116). Alpha-enolase, a critical glycolytic protein, interconverts 2-phosphoglycerate to phosphoenolpyruvate (Figure 10.8). The enzymatic activity of alpha-enolase is reduced significantly compared to control samples (83). Reduction in enzymatic activity can lead to protein dysfunction and in this case, lowered ATP production.

FIGURE 10.8

Interconversion of 2-phosphoglycerate to phosphoenolpyruvate via enolase.

Pyruvate kinase is also a glycolytic enzyme that catalyzes the final step in glycolysis, the conversion of phosphoenolpyruvate to pyruvate with the concomitant transfer of the high-energy phosphate group from phosphoenolpyruvate to ADP, thereby generating ATP (Figure 10.9). Under aerobic conditions, pyruvate can be transported to the mitochondria, where it enters the TCA cycle and is further broken down to produce considerably more ATP through oxidative phosphorylation. Therefore, the oxidative inactivation of this important enzyme in the hippocampi of MCI subjects could conceivably result in the reduced ATP production and alter ATP-dependent processes, such as signal transduction and cell potential maintenance, thereby leading to altered Ca²⁺ homeostasis and neuronal dysfunction.

FIGURE 10.9

Pyruvate kinase enzymatic reaction.

Lactate dehydrogenase B (LDH) reduces pyruvate to lactate by NADH (Figure 10.10). Lactate is a substrate for gluconeogenesis and since glucose is the major supplier of energy to the brain, proper lactate production is crucial (117). LDH dysfunction and subsequent reduced glucose metabolism are commonly observed in the positron emission tomography (PET) scans of MCI and AD brain (118,119). Enzyme activity of lactate dehydrogenase is significantly reduced in MCI hippocampus, which further correlates protein dysfunction and enzyme activity impairment. Likewise, specific enzyme activity was also lower in MCI hippocampus. Impairment of this enzyme could initiate reduction of glucose production and creation of excess pyruvate.

FIGURE 10.10

Conversion of pyruvate to L-lactate via lactate dehydrogenase.

Phosphoglycerate kinase catalyzes the reaction to convert 1,3-bisphosphoglycerate to 3-phosphoglycerate. This reaction undergoes substrate phosphorylation by phos-phoryl transfer from 1,3-bisphosphoglycerate to ADP to produce ATP (Figure 10.11). Additionally, enzyme activity is reduced, thus suggesting that oxidative modification leads to impairment of protein function. Impairment of phosphoglycerate kinase results in decreased energy production and irreversible downstream effects, such as multidrug resistance (120).

FIGURE 10.11

Phosphoglycerate kinase enzymatic reaction.

Aldolase is an essential protein that cleaves fructose 1,6-bisphosphate to dihydroxy-acetonephosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) (Figure 10.12). The production of G3P is essential in continuing glycolysis in order to produce adequate amounts of ATP to maintain ion-motive ATPases, signal transduction, glu-tamate, and glucose transporters (121,122). Oxidative modification of aldolase can compromise membrane symmetry and render neurons vulnerable to excitotoxicity and apoptosis.

FIGURE 10.12

Aldolase enzymatic reaction.

The impairment of these energy-related proteins would lead to decreased ATP production, with consequent dysfunction in electrochemical gradients, ion pumps, and voltage-gated ion channels, glucose and glutamate transporters, loss of membrane asymmetry, as well as lower efficiency on such processes as Ca²⁺ homeostasis, cell potential, and signal transduction. All these glycolytic protein oxidative modifications observed in the earliest stage of AD support the hypothesis of energy metabolism alteration in Alzheimer’s disease and are consistent with the known PET alterations in MCI and AD.

10.11. NEUROPLASTICITY

It is well documented that glutamine synthetase (GLUL) activity declines in AD (103,123–125). GLUL catalyzes the rapid amination of glutamate to form the non-neurotoxic amino acid glutamine. This reaction maintains the optimal level of glutamate and ammonia in neurons and modulates excitotoxicity. Together with the action of glutamate receptors and glutamate transporters, this process is important to maintaining neuroplasticity (126). A decline in neuroplasticity is suggested to correlate with the progression of AD from MCI (127). Therefore, oxidative inactivation of GLUL suggests the glutamate-glutamine cycles in hippocampi of MCI subjects are impaired, which may contribute to excitoxicity and impairment of neuroplasticity during the development of AD (128).

10.12. MITOCHONDRIAL DYSFUNCTION

Mitochondrial dysfunction is associated with AD; therefore, the proteomic identification of ATP synthase in MCI IPL and hippocampus contributes to evidence suggesting a role of mitochondrial dysfunction in the progression of AD. ATP synthase alpha-chain is a mitochondrial regulating subunit of complex V, which plays a key role in energy production. Alteration of complex V of the electron transport chain, ATP synthase, results in impaired ATP production in the mitochondria, the ATP “powerhouse.” ATP synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha and beta) to produce ATP. ATP synthase, alpha-subunit, is the only HNE-modified protein observed by redox proteomics in both MCI hippocampus and IPL. At an early stage, ATP synthase is tightly associated with tau aggregated proteins in neurofibrillary tangles in AD (129), making ATP synthase a potential target for AD therapeutics. The oxidation of ATP synthase leads to the inactivation of this mitochondrial complex. Failure of ATP synthase could contribute to a decrease in the activity of the entire electron transport chain and impaired ATP production, resulting in possible electron leakage from their carrier molecules to generate ROS, suggesting an alternative rationalization for the well-documented existence of oxidative stress in AD and MCI (24,26,31,32,83,130,131). The specific activity of ATP synthase was reduced in MCI hippocampus and IPL compared to age-matched controls, providing insight into enzyme efficiency in MCI (28). Additionally, malate dehydrogenase is oxidatively modified in MCI; this enzyme is responsible for oxidation of L-malate to oxaloacetate in the citric acid cycle, another ATP-producing metabolic pathway (Figure 10.13).

FIGURE 10.13

Malate dehydrogenase enzymatic reaction.

As chaperones, heat shock proteins assist in establishing proper protein conformation in an effort to prevent protein aggregation by repairing misfolded proteins or guiding misfolding proteins to the proteosome for degradation (132). Numerous heat shock proteins (Hsp70, Hsc71, and Hsp90 and 60, respectively) have been found to be oxidatively modified in neurodegenerative diseases including MCI (133), AD (112), and HD (134). Oxidative modification of heat shock proteins may exacerbate protein misfolding and protein aggregation, leading to reduced effective proteosomal activity and eventual proteosomal overload and dysfunction, known to occur in AD (135).

10.13. ANTIOXIDANT DEFENSE

Peptidyl-prolyl cis/trans isomerase (Pin-1) is a regulatory protein that reversibly alters the conformation of proline residues in proteins from cis to trans (136). The WW domain of Pin-1 recognizes phosphorylated Ser-Pro and phosphorylated Thr-Pro motifs in proteins, and thereby binds to many cell cycle-regulating proteins, APP, and tau protein. Pin-1 is co-localized with phosphorylated tau and also shows an inverse relationship to the expression of tau in AD brains (137–139). Moreover, Pin-1 is oxidatively inhibited in AD brain (140) and is able to restore the function of tau protein in AD (141). Therefore, the oxidative inactivation of Pin-1 in the hippocampi of MCI subjects could be one of the initial events that trigger tangle formation, oxidative damage, cell cycle alterations, and eventual neuronal death in AD brains. Pin-1 specifically recognizes pSer-Pro and pThr-Pro motifs. Pin-1 is excessively carbonylated in MCI (83,142) and AD hippocampus (104). Moreover, Pin-1 binds to APP and affects production of amyloid beta peptide. Pin-1 enzymatic activity is significantly decreased in MCI and AD brain, contributing to previous research showing loss of enzymatic activity in oxidatively modified proteins (103,143). This decrease in Pin-1 activity may be responsible for the increased accumulation of both phosphorylated tau protein in AD hippocampus, which is rich in neurofibrillary tangles (NFT) and senile plaques (SP), rich in amyloid beta.

Glutathione S-transferase mu and multidrug resistance protein 3 are nitrated in MCI IPL. These two proteins play an important role in regulating cellular processes by decreasing the levels of oxidants or by removing toxic compounds that are generated in the cell. Glutathione S-transferases (GSTs) catalyze the binding of HNE to glutathione, resulting in the formation of a GSH-HNE conjugate that is exported out from the cell by the multidrug resistant protein-1 (MRP1). This process thereby plays an important role in cellular protection against oxidative stress. In AD brain, GST protein levels and activity were reported to be decreased; in addition, GST was found to be oxidatively modified by HNE (144). GSTs have a high catalytic activity against HNE and are oxidatively modified and downregulated in AD brain (144). As a corollary, overexpression of GST can combat the effects of HNE toxicity in culture (145).

Peroxiredoxin 6 (Prx VI) is found to be nitrated in MCI hippocampus. Peroxiredoxins remove toxic hydrogen peroxide from the cell, resulting in reduced ROS production. Peroxiredoxins can reduce peroxynitrite at a high catalytic rate, which may modulate protein nitration and cell damage (146). Prx VI is the only peroxiredoxin that uses glutathione as an electron donor, while all other peroxiredoxins (PrxI-PrxV) use thioredoxin. In addition, peroxiredoxins play a role in cell differentiation and apoptosis. The decrease in the activity of this enzyme may also lead to decreased phospholipase A2 activity, one of the target proteins regulated by peptidyl prolyl cis/trans isomerase (Pin-1), a protein that has been reported to be downregulated and have decreased activity in MCI and AD brain (104,141,142).

The enzyme GST forms a complex with Prx VI in order to modulate both enzyme activities. These proteins work in coordination with one another either directly or indirectly, thereby protecting the cell from toxicants. These results provide insight into how the changes of these proteins may contribute to tau hyperphosphorylation and neurofibrillary tangle formation, in addition to development of oxidative stress. This result could conceivably be related to the identification of MRP1 as a protein with elevated HNE binding in AD (144). This protein was also mapped to undergo DNA damage by oxidative stress mechanisms (147).

Carbonyl reductase is a vital enzyme that can reduce carbonyl-containing compounds to their resultant alcohols, thereby reducing protein carbonyl levels. Subsequent malfunction or downregulation of this enzyme could cause an increase in protein carbonyls, which because of the polarity of the carbonyl moiety could expose ordinarily buried hydrophobic amino acids to the solvent (i.e., disrupt conformation). Carbonyl reductase has been shown to reduce the lipid peroxidation product, HNE (148). Carbonyl reductase expression is altered in Down’s syndrome and AD patients (149). The gene for carbonyl reductase is located in close proximity to the gene for Cu/Zn superoxide dismutase (SOD1) (150). Interestingly, the genes for SOD1, carbonyl reductase, and APP are located on chromosome 21, which is a trisomy in Down’s syndrome patients (151). The link between increased amyloid deposition and decreased carbonyl reductase enzymatic activity is unclear. The current research posits a possible intriguing relationship among amyloid beta, Down’s syndrome, and carbonyl reductase in neurodegeneration.

Oxidative stress is the imbalance of pro-oxidants and antioxidants, where equilibrium is favoring the side of pro-oxidants. This can also mean antioxidant defense is low due to protein modification. Increased protein expression of peroxiredoxins (I and II), Cu/ZnSOD, carbonyl reductase, and alcohol dehydrogenase, glutathione S-transferase (GST), and multidrug resistant protein-1 (MRP1) in AD brain has been reported (149,152–155). Similarly, antioxidant defense is altered in MCI (156). Several antioxidant proteins undergo oxidative modification in MCI brain (133).

10.14. STRUCTURAL DYSFUNCTION

Dihydropyriminidase related protein 2 (DRP-2) and fascin 1 are identified as nitrated proteins in MCI hippocampus. DRP-2 is a member of the dihydropyrimidinase-related protein family that is involved in axonal outgrowth and pathfinding through transmission and modulation of extracellular signals (157,158). Additionally, DRP-2 interacts with and modulates collapsin, which aids in dendrite elongation, guidance, and growth cone collapse. DRP-2 has been reported to be associated with NFT, which may lead to decreased levels of cytosolic DRP-2 in AD (159). This, in turn, would eventually lead to abnormal neuritic and axonal growth, thus accelerating neuronal degeneration in AD (160), which is one of the characteristic hallmarks of AD pathology. Increased oxidation (41) and decreased expression of DRP-2 protein was observed in AD. In adult Down’s syndrome (DS) (159), fetal DS (161), schizophrenia, and affective disorders, DRP-2 has lower levels in brain. Since memory and learning are associated with synaptic remodeling, nitration and subsequent loss of function of this protein could conceivably be involved in the observed memory decline in MCI. Moreover, the decreased function of DRP-2 could be involved in the shortened dendritic length and synapse loss observed in AD (162). Maintaining neuronal communication is essential in learning and memory. Since memory is greatly depreciated in MCI (163,164), certain proteins involved in neuronal communication are oxidatively modified in MCI and AD (112,133,155).

Fascin 1 (FSCN1) is an actin-bundling, structural protein also known as p55 (165) and is involved in cell adhesion (166) and cell motility (167). It is a marker for dendritic functionality (168). Addition of p55 has been shown to protect cells from oxidative stress produced by an insult (169). The identification of this protein as nitrated in MCI brain is consistent with the notion that loss of function of this protein lessens protection against oxidative damage and could be an important event in the transition of MCI to AD. Fascin 1 has also been found to interact with protein kinase C alpha (PKCα), which regulates focal adhesions (170). Impairment of this protein can be related to faulty neurotransmission from the affected dendritic projections. In a beagle dog model of AD, in which beagle amyloid beta has the same sequence as human amyloid beta, fascin was a protein protected by a diet rich in antioxidants and in a program of environmental enrichment, i.e., making new synapses (171).

Actin is a principal protein playing a central role in maintaining cellular integrity, morphology, and the structure of the plasma membrane. Actin microfilaments play a role in the neuronal membrane cytoskeleton by maintaining the distribution of membrane proteins, and segregating axonal and dendritic proteins (172). In the CNS, actin is distributed widely in neurons, astrocytes, and blood vessels (173) and is particularly concentrated in presynaptic terminals, dendritic spines, and growth cones. Oxidation of actin, by HNE modification, can lead to loss of membrane cytoskeletal structure, decreased membrane fluidity, and trafficking of synaptic proteins and mitochondria. Moreover, actin is involved in the elongation of the growth cone, and loss of function of actin could play a role in synapse loss and neuronal communication, which may be associated with progressive memory loss documented in AD (174).

10.15. SIGNAL TRANSDUCTION

14–3–3-protein gamma is found to be nitrated in MCI IPL. 14–3–3 gamma is a member of the 14–3–3 protein families, which are highly expressed in the brain (175,176). These proteins are involved in a number of cellular functions including signal transduction, protein trafficking, and metabolism (175). 14–3–3 gamma is associated with neurofibrillary tangles in AD (177) and levels of 14–3–3 proteins are increased in MCI brain (155), AD brain (178,179), AD CSF (180), calorically restricted rats (181), and ICV and neuronal models of AD (116,182). The nitration of 14–3–3 gamma could change its conformation, which conceivably could lead to altered binding to two of its normal binding partners, glycogen synthase kinase 3 beta (GSK3ß) and tau. One of the isoforms of 14–3–3 can act as a scaffolding protein and simultaneously bind to tau and GSK3ß in a multiprotein tau phosphorylation complex (183). This complex may promote tau phosphorylation and polymerization (184,185), leading to the formation of tangles and further leading to neurodegeneration in AD.

Neuropolypeptide h3 is critical for modulation of the enzyme choline acetyltransferase, which is vital in signal transduction and cell communication. The loss of choline acetyltransferase leads to reduced levels of the neurotransmitter acetylcholine, causing poor neurotransmission (186). NMDA receptors activate the production of this enzyme, and alteration of the NMDA receptor mediates cholinergic deficits (187). AD has cholinergic deficits, consistent with dysregulation in acetylcholine levels and loss of cholinergic neurons (188–191). Consequently, the oxidative modification of this protein in MCI and AD (113) highlights the role of cholinergism in the development of this dementing disease.

10.16. PROTEIN SYNTHESIS

EF-Tu and eIF-alpha are intimately involved in protein synthesis machinery. Oxidation, and possibly impairment, of initiation factors of protein synthesis in MCI patients were shown by redox proteomics (28). Human mitochondrial EF-Tu (EF-Tu) is a nuclear-encoded protein and functions in the translational apparatus of mitochondria (192). Like its prokaryotic homolog, the mammalian EF-Tu GTPase hydrolyzes a molecule of GTP each time an amino-acylated tRNA is accommodated on the A site of the ribosome, and its recycling depends on the exchange factor EF-Ts. Nuclear genes encode most respiratory chain subunits and all protein components necessary for maintenance and expression of mtDNA. Mitochondria play pivotal roles in eukaryotic cells in producing cellular energy and essential metabolites as well as in controlling apoptosis by integrating numerous death signals (193). Mitochondrial protein synthesis inhibition is associated with the impairment of differentiation in different cell types, including neurons (194). The coordination of mitochondrial and nuclear genetic systems in the cell is necessary for proper mitochondrial biogenesis and cellular functioning. eIF-α is an abundant protein required to bind aminoacyl-tRNA to acceptor sites of ribosomes in a GTP-dependent manner during protein synthesis (195). Recently, eIF-α has been shown to be involved in cytoskeletal organization by binding and bundling actin filaments and microtubules. eIF-α is an important determinant of cell proliferation and senescence (196), since it is regulated in aging, transformation, and growth arrest. Inhibition of eIF-α induces apoptosis (197), indicating that eIF-α activity is critical to normal cell function. Numerous studies have provided indirect evidence that suggests alterations in protein synthesis may occur in AD (53,198–201) and decreased protein synthesis in AD and MCI (53,202). The dysfunction of the protein synthesis apparatus, mediated in part by oxidative stress, could compromise the ability of cells to generate the various factors needed to regulate cell homeostasis, thus contributing to impaired neuronal function and to the development of neuropathology in MCI patients.

10.17. CONCLUSIONS

In conclusion, the redox proteomics-identified brain proteins in MCI brain play important roles in different neuronal functions and are directly or indirectly linked to AD pathology. Comparative analysis of these proteins between MCI IPL and hippocampus brain regions showed alpha enolase as a common target of protein oxidation by all three modifications mentioned. This suggests that energy metabolism may be among the first cellular properties that become severely affected in MCI. A similar sensitivity to energy metabolism was observed in AD brain (112,113,116,203). Future studies using animal models of the different stages of this dementing disorder should help in further delineating the mechanisms of MCI pathogenesis and to develop effective therapies to combat conversion of MCI to AD. Several HNE-bound and nitrated proteins identified in MCI were identical to those found in not only AD, but other neurodegenerative diseases. These enzymes are involved in cholinergic processes, energy metabolism, transport, detoxification, protein synthesis, and stress response properties. Loss of function or modification to any protein can be catastrophic for neuronal communication, ATP production, and other essential cell functions. Because oxidative stress is involved in arguably the earliest phase of AD (31,32,83), and because MCI may be considered a prodromal phase of AD, the identification of specific target proteins that are oxidatively modified by products of lipid peroxidation and protein nitration may provide vital insights into the role of oxidative damage in mechanisms of neuronal death in AD. The current work forms a framework for subsequent experiments and provides potential new targets for neuroprotective therapeutic intervention in MCI.

ACKNOWLEDGMENTS

The authors thank the University of Kentucky A.D.C. Clinical and Neuropathology Cores for providing the brain specimens used for this study. This research was supported in part by grants from NIH (AG-10836; AG-05119; AG-029839).

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