3.Carbonylated proteins in brain of subjects with amnestic MCI
In brain from subjects with amnestic MCI compared with age-matched controls, CA II, Hsp70, mitogen-activated protein kinase I (MAPKI), syntaxin binding protein I (SBP1), Eno1, GS, pyruvate kinase M2, and Pin1 showed significant increased carbonylation. CA II, Eno1, GS, and Pin1were discussed just now in the context to AD pathology or clinical presentation, and similar considerations apply to amnestic MCI.
Hsp70 is neuroprotective against intracellular Aβ; however, this protein is carbonylated in AD, thereby reducing its cellular protection (264
). Several other HSPs have been found to be oxidatively modified in AD (74
), including Hsp90 and Hsp60 (123
), while Hsp 27 and Hsp 32 levels are elevated in amnestic MCI (123
). Impairment of these proteins could contribute to proteasomal overload and dysfunction, observed in AD (221
). Aβ-treated synaptosomes show that HSPs are oxidatively modified (50
), further illustrating the vulnerability of HSPs to Aβ-induced OS.
Pyruvate kinase catalyzes the final step in glycolysis, the conversion of phosphoenolpyruvate to pyruvate with the concurrent transfer of the phosphate group from phosphoenolpyruvate to ADP, thereby generating ATP. Under aerobic conditions, pyruvate can be transported to the mitochondria, where it is converted to acetyl coenzyme A, the latter entering the tricarboxylic acid (TCA) cycle and further metabolic processes that produce considerably more ATP through oxidative phosphorylation. Anaerobically, pyruvate can be reduced to lactate. Additionally, enzymatic activity is reduced, thus suggesting that oxidative modification leads to loss of protein function. Considerations just given for loss of ATP and altered PET scans in AD also apply to MCI subjects.
SBP1 is a neuron-specific protein that binds strongly to syntaxin 1 and is important for synaptic vesicle exocytosis and neurotransmitter release, a key process for neurotransmission. As just discussed, oxidation leads to loss of function of SBP1, which could impair neurotransmission and subsequently might contribute to loss of neuronal function, eventually leading to loss of memory and cognition and neurodegenerative processes involved in progression of MCI to AD.
Recent studies suggested mitogen-activated protein kinases (MAPKs) as key regulators of the formation of plaques and tau hyperphosphorylation in AD (147
). MAPKs pathways transduce intracellular signaling to increase expression of different proteins; dysregulation of MAPK-dependent pathways suggests a systematic disorder of protein translation regulation in MCI brains. ERK activation is present in EAD astroglia, while in more advanced AD, it is associated with neuronal cell bodies and dystrophic neurites around plaques, suggesting that ERK activation in astroglia may be an important early response to the onset of AD pathology (169
). More recently, abnormal phosphorylation of tau was reported to correlate with increased activity of ERK1/2 in postmortem AD brains (300
). Oxidative modification of MAPKs might make them more prone to phosphorylation or may be an alternative mechanism of their activation, thus initiating signaling cascades, ultimately leading to hyperphosphorylation of tau. Based on the existing literature, we hypothesize that amyloid-induced oxidation of MAPK might contribute to increased phosphorylation of tau in AD, leading to cell death.
6.Protein-bound HNE in brain and progression of Alzheimer's disease
As just discussed, HNE is a reactive product of lipid peroxidation, and this α,β-unsaturated alkenal binds to Cys, His, or Lys residues of proteins, thereby changing the conformation and function of proteins (66
). In AD, HNE has been found to be significantly elevated in AD brain, plasma, and CSF (187
). PCAD subjects have clinically normal antemortem psychometric scores but brain pathology that meets the neuropathological criteria for AD and exhibit no significant brain cell loss or neuronal atrophy (202
). Although no alteration of protein-bound HNE was found in PCAD IPL, increased levels of total HNE and acrolein in hippocampus were reported (51
). This section of this comprehensive article deals specifically with the HNE modifications observed in the other three progressive stages of AD: MCI, EAD, and LAD.
In amnestic MCI, several proteins have been identified by redox proteomics as HNE-conjugated in the hippocampus and IPL brain regions. These proteins include Eno1, phosphoglycerate kinase, lactate dehydrogenase B, pyruvate kinase, ATP synthase, neuropolypeptide h3, HSP70, CR1, β-actin, initiation factor alpha, and elongation factor Tu (EF-Tu) (320
). Since altered energy metabolism and reduced cholinergic activity are two well-documented hypotheses of AD, the HNE modification of several cholinergic, glycolytic, and ATP generating proteins support the notion of involvement of these pathways in AD.
ATP synthase, Eno1, and pyruvate kinase have been just discussed and also have been found to be oxidatively modified in AD brain. Another glycolytic enzyme that is found to be HNE modified in MCI brain is phosphoglycerate kinase, which catalyzes the dephosphorylation of 1,3-bisphosphoglycerate to 3-phosphoglycerate. This reaction undergoes substrate-level phosphorylation by phosphoryl transfer from 1,3-bisphosphoglycerate to ADP to produce one molecule of ATP. Impairment of this glycolytic enzyme results in decreased energy production and irreversible downstream effects, such as multidrug resistance (132
). This result could conceivably be related to the identification of multidrug resistant protein 1 (MRP1) as a protein with elevated HNE binding in AD (364
Lactate dehydrogenase B anaerobically reduces pyruvate to lactate through lactic acid fermentation using NADH as a cofactor. The NAD+
generated in this process is used in glycolysis to oxidize G3P to 1, 3-bisphosphoglycerate, an important reason for this reaction. Lactate is a substrate for gluconeogenesis and given that glucose is the major supplier of energy to the brain, proper lactate production is crucial (223
). Lactate dehydrogenase enzymatic activity is significantly reduced in MCI hippocampus (320
), which provides supplemental evidence for the correlation between protein dysfunction and enzyme activity impairment. Dysfunction of this enzyme could yield excess pyruvate and a reduction in the production of glucose.
Actin is a principal protein that plays a central role in maintaining structural integrity, cell morphology, and 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 (33
). In the CNS, actin is distributed widely in neurons, astrocytes, and blood vessels. It is particularly concentrated in growth cones, dendritic spines, and presynaptic terminals. HNE conjugation of actin 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 the loss of synapse and neuronal communication documented in AD (267
CR is an enzyme that reduces carbonyl-containing compounds to their resultant alcohols, thereby reducing the level of PCO. Subsequent malfunction or downregulation of this enzyme would be consistent with increased PCO, which, because of the polarity of the carbonyl moiety, could expose normally buried hydrophobic amino acids to the protein surface, resulting in a disruption of conformation. CR has been shown to reduce the lipid peroxidation product, HNE (129
). CR expression is altered in DS and AD patients (26
). This enzyme was found to be modified in persons with corticobasal degeneration, a neurological disorder whose symptoms closely mirror that of PD (100
). The gene for CR is located in close proximity to the gene for the antioxidant enzyme, Cu/ZnSOD (244
). Interestingly, the genes for SOD1, CR, and APP are located on chromosome 21, which is a trisomy in DS patients (154
). A potential link between DS and AD by irregular meiotic recombination in chromosome 21 (308
) has been postulated. Current research supports a possible relationship among CR, DS, and Aβ in neurodegeneration.
EF-Tu and eukaryotic initiation factor α (eIF-α) are intimately involved in protein synthesis machinery. Human mitochondrial EF-Tu is a nuclear-encoded protein and functions in the translational apparatus of mitochondria. Mammalian EF-Tu acts as a GTPase by hydrolyzing one molecule of GTP for each A site amino-acylated tRNA of the ribosome. As just discussed, mitochondria play pivotal roles in eukaryotic cells in producing cellular energy and essential metabolites as well as in controlling apoptosis by integrating various death signals (294
). Mitochondrial protein synthesis inhibition, either by deleting mtDNA or by blocking translation in the organelle, is associated with the impairment of differentiation in different cell types, including neurons (390
). Loss of neuronal differentiation can lead to an incomplete development of the neuron, which would result in reduced neurotransmission.
eIF-α, which binds aminoacyl-tRNA to acceptor sites of ribosomes in a GTP-dependent manner (306
), is involved in cytoskeletal organization by bundling and binding actin filaments and microtubules. The expression level of eIF-α is regulated in aging, transformation, and growth arrest. Due to eIF-α regulation in differing states of cell life and its key position in protein synthesis and cytoskeletal organization, this protein is an important determinant of cell proliferation and senescence (379
). Inhibition of eIF-α promotes apoptosis (306
), indicating that eIF-α activity is critical to normal cell function.
Taken together, increased levels of HNE-bound eIF-α and EF-Tu suggest an impairment of protein synthesis machinery, either in cytosol or mitochondria, associated with an impairment of the rate and specificity of ribosome functions. Numerous studies have provided indirect evidence that suggests alterations in protein synthesis may occur in AD (128
). The dysfunction of the protein synthesis apparatus, mediated in part by redox proteomics identified oxidatively dysfunctional EF-Tu and eIF-α, could compromise the ability of brain cells to generate the countless factors needed to regulate cell homeostasis, thus contributing to impaired neuronal function and to the development of neuropathology in AD.
Neuropolypeptide h3 is critical for modulation of choline acetyltransferase, an enzyme essential in the synthesis of acetylcholine. The loss of choline acetyltransferase leads to reduced levels of acetylcholine, causing poor neurotransmission (291
). NMDA receptors activate the production of this enzyme, and modulation of the NMDA receptor mediates cholinergic deficits (213
). AD patients have considerable cholinergic deficits, consistent with dysregulation of acetylcholine levels and loss of cholinergic neurons (328
). The oxidative modification of this protein further supports the involvement of cholinergic neurons in AD, an early hypothesis of this disorder (156
). Neuropolypeptide h3 undergoes HNE modification in MCI hippocampus and nitration in late-stage AD (68
). Neuropolypeptide h3 has several other names including phosphatidylethanolamine binding protein (PEBP), hippocampal cholinergic neurostimulating peptide, and Raf kinase inhibitor protein (RKIP). As a PEBP, PEBP may be important in phospholipid asymmetry. Apoptosis is initiated when phosphatidylserine resides on the outer leaflet of the membrane. Loss of function and changes in conformation of PEBP conceivably could lead to loss of phospholipid asymmetry, a signal for neuronal apoptosis, which further supports the role of PEBP as a parapoptosis inhibitor (359
). Loss of PEBP may impact lipid asymmetry, as loss of activity is observed in AD and MCI and mouse models of familial AD (23
) and can potentially disrupt cellular homeostasis. PEBP levels are decreased in AD, which promotes amyloid beta accumulation in the Tg2576 transgenic mouse model of AD (166
). RAF kinases are serine/threonine protein kinases involved in cell signaling in the mitogen-activated protein cascade and NF-kappa B. RKIP disrupts this signaling pathway by interacting with RAF1-MEK 1/2 and NF-kappa B inducing kinase, causing the inhibition of NF-kappa B activation and regulating apoptosis. As demonstrated by the various functions through its numerous monikers, neuropolypeptide h3 is a highly important protein and oxidative modification is likely detrimental to neurons.
EAD, as just mentioned, is thought to be a transitional stage of AD in which patients exhibit progressive cognitive deficits and display mild dementia on clinical evaluation. Redox proteomics analysis identified two HNE-conjugated proteins in this stage of AD that overlap those in the preceding stage of AD, MCI. These proteins include Eno1 and ATP synthase, which were just discussed. Additionally, triose phosphate isomerase, malate dehydogenase, MnSOD, and DRP2 (CRMP2) undergo HNE conjugation in EAD brain as identified by redox proteomics (322
Oxidative impairment of mitochondrial resident MnSOD is likely a contributing factor to the mitochondrial dysfunction associated with AD. Activity for MnSOD is significantly reduced in EAD brain and CSF compared with the age-matched control, which is consistent with the concept of mitochondrial dysfunction as a factor in the progression of AD. MnSOD was also found to be nitrated and subsequently inactivated in mice by peroxynitrite (121
). Overexpression of SOD2 increases Aβ degradation, while partial deficiency promotes Aβ deposition, thereby likely contributing to cognitive decline observed in a transgenic mouse model of AD (134
Malate dehydrogenase (MDH) catalyzes the reversible oxidation of malate to oxaloacetate by NAD+
in the TCA cycle. MDH links glycolysis to the ETC by transferring NADH to NADH dehydrogenase (Complex I) through the malate-aspartate shuttle resulting in the production of ATP. In contrast to elevated HNE binding to MDH, MDH levels were increased in AD patients, but the level of protein oxidation of MDH was not significant, which probably highlights a compensatory mechanism in response to OS (234
). Activity of MDH increases during aging (58
), which further supports the hypothesis of mitochondrial dysfunction in AD.
In late-stage AD, redox proteomics identified four HNE-modified proteins that overlap EAD (Eno1, ATP synthase, MnSOD, and CRMP2) (304
). Both Eno1 and ATP synthase are consistently HNE modified in all transitional stages of AD, providing evidence for the altered energy metabolism and mitochondrial dysfunction hypotheses associated in the progression of AD (66
). Other HNE-modified brain proteins in AD were identified by redox proteomics (304
): Aldolase (ALDO1) cleaves fructose 1,6-bisphosphate and produces the two glycolytic intermediates, G3P and DHAP. Fructose 1,6-bisphosphate is neuroprotective and preserves GSH in cortical neurons during OS conditions (391
). ALDO1 catalyzes a critical step, as it generates two substrates that are used to eventually produce 2 molecules of ATP and more in TCA and ETC chain. Consequently, HNE modification results in decreased energy metabolism. Levels of ALDO1 are significantly decreased in AD hippocampus (41
) and PD (173
). Enzyme activity is reduced (41
), and impairment can cause increased levels of fructose 1,6-bisphosphate, inhibition of complete glycolysis, and ATP depletion.
Aconitase catalyzes the isomerization of citrate to isocitrate in the TCA cycle. As an iron-sulfur protein, its Fe-S cluster participates in the hydration—dehydration reaction that occurs. The three cysteine residues in the Fe-S core can undergo Michael addition and form acrolein, HNE, and maldondialdehyde conjugated adducts, thereby increasing lipid peroxidation markers (248
). Enzymatic activity of this enzyme is significantly reduced in AD, thereby yielding in protein dysfunction (304
). The TCA cycle takes place in the mitochondria; therefore, aconitase impairment results in mitochondrial dysfunction, a common theme of neurodegenerative diseases (405
). As just noted, decreased ATP production can lead to voltage-gated channel and ion-motive pump disruption as well as synapse loss, an early event in Alzheimer's disease pathology (335
α-Tubulin is an isoform of tubulin that alternates with β-tubulin to form a prominent cytoskeletal structure, the microtubule. Microtubules are used to transport cargo (i.e.
, vesicles and organelles) from the cell body to the periphery and vice versa. On HNE modification, α-tubulin is structurally altered and microtubules depolymerize (162
). Therefore, cargo cannot reach their destination and the cytoskeleton is altered (284
). This could contribute to the notion that synaptic domains are the first to be damaged in AD neurons (235
Prxs are a family of antioxidant enzymes that are pivotal in antioxidant defense as just discussed. PRX VI is a 1-Cys Prx that plays a role as a second messenger for growth factors and cytokines. Prx VI, a GPx that exhibits Ca2+
-independent phospholipase A2
), is cytosolic and is expressed in astrocytes and in neurons at low levels (94
). In addition, the decrease in the activity of this enzyme may also lead to decreased phospholipase A2
activity. Phospholipase A2
is a target for regulation by Pin1, which as just discussed, has been reported to be downregulated and showed oxidative dysfunction in the AD brain (65
). PRX VI has been found to be protective against mitochondrial dysfunction, a feature that pinpoints its effectiveness as an antioxidant (136
). PRX VI also plays important roles in cell differentiation and apoptosis, and HNE modification may lead to tau hyperphosphorylation and NFT formation in addition to development of OS.
8.Nitrated brain proteins in MCI
Eno1, glucose-regulated protein precursor, ALDO1, glutathione-S
-transferases (GST) Mu, multidrug resistant protein 3 (MRP3), 14-3-3 protein gamma, MDH, PR VI, DRP-2 (CRMP2), Fascin 1 (FSCN1), and HSPA8 protein were identified as nitrated proteins in MCI by redox proteomics (372
). Most of these proteins also were found to be oxidatively modified in AD, and have been discussed pertaining to AD.
The brain proteins that are found nitrated in MCI but not in AD includeGST Mu, MRP3, 14-3-3 protein gamma, and FSCN1. One of the mechanisms for the removal of toxic metabolites from cells is accomplished via
GST and MRP proteins. GST conjugates HNE to GSH, resulting in the formation of GS-HNE adducts that are effluxed out of cells via
MRP efflux pumps. Hence, oxidation and functional impairment of these proteins would lead to increased accumulation of HNE in the cell and, consequently, in cell death. In the MCI brain, increased levels of protein-bound HNE have been found (78
). In the AD brain, GST protein levels and activity were reported to be decreased; in addition, GST was found to be oxidatively modified by HNE (364
14-3-3-protein gamma is a member of the 14-3-3 protein family. These proteins are involved in a number of cellular functions including signal transduction, protein trafficking and metabolism. In the AD brain (239
), CSF (60
), and ICV animal model of AD (158
), the levels of 14-3-3 proteins are increased, which conceivably could lead to altered binding to two of its normal binding partners, glycogen synthase kinase 3 and tau, and may promote tau phosphorylation and polymerization, conceivably contributing to the formation of tangles and subsequent neurodegeneration in AD.
FSCN1, also known as p55, is a structural protein involved in cell adhesion and cell motility (403
). P55 protects cells from OS and is used as a marker for dendritic functionality. FSCN1 was also shown to interact with protein kinase C (16
), thereby playing important rules in post-translational protein modification. Impairment of this protein is conceivably related to faulty neurotransmission from the affected dendritic projections, to altered intracellular signaling, and may contribute to the progression of AD.
9.Nitrated proteins in EAD
Protein nitration is increased in EAD subjects compared with age-matched controls (321
). In the EAD brain, redox proteomics analysis identified the increased nitration of Prx2, TPI, glutamate dehydrogenase, neuropolypeptide h3, PGM1, H-transporting ATPase, Eno1, and ALDO1 (321
). All these proteins were identified as either the target of protein carbonylation or HNE modification and have been just discussed.
In summary, redox proteomics analyses of brain proteins throughout the spectrum of AD have identified proteins whose oxidative dysfunction is consistent with the clinical presentation, pathology, and/or biochemistry of this disorder, demonstrating the power and utility of this technique. Oxidative dysfunction of proteins involved in ATP production, excitotoxicity, detoxification, protein degradation, neuritic abnormalities, and mitochondrial abnormalities are likely involved in neurodegeneration at various stages of this dementing disorder. Taken together, the redox proteomics studies in amnestic MCI, EAD, and late-stage AD identified Eno1 as the common target of protein carbonylation, HNE modification, and nitration between AD, EAD, and MCI, consistent with the notion that this protein should be critical to AD progression and pathogenesis. , , and show the common targets of protein carbonylation, HNE, modification and nitration, respectively, among AD, MCI, and EAD. The identification of these common targets of protein oxidative modification among different stages of disease is consistent with the concept that losses of function of these proteins are key in the progression and pathogenesis of AD. Continued studies are in progress in our laboratory to understand the role of oxidatively modified proteins in AD pathogenesis.
FIG. 13. Venn diagram of HNE-modified proteins identified during the progression of AD. Alpha-enolase and ATP synthase are the common targets of oxidative modification between AD, MCI, and EAD, and oxidative modification of these proteins might be key in the progression (more ...)
FIG. 14. Venn diagram of excessively carbonylated proteins throughout the pathogenesis of AD. Enolase, ATP synthase alpha, and UCH-L1 are the common targets of oxidation between AD, MCI, EAD, and PCAD. PCAD, preclinical Alzheimer disease; UCH-L1, ubiquitin carboxy-terminal (more ...)
FIG. 15. Nitrated proteins identified during the progression of AD in the hippocampus1 and IPL2 regions. DRP2 and aldolase are common targets of nitration between MCI and EAD. The identification of α-enolase as the only common target of protein nitration (more ...)