Proteomic identification of nitrated proteins in Alzheimer's disease brain

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.     

Protein glycation, oxidation and nitration adduct residues and free adducts of cerebrospinal fluid in Alzheimer's disease and link to cognitive impairment

Increased damage to proteins by glycation, oxidation and nitration has been implicated in neuronal cell death leading to Alzheimer's disease (AD). Protein glycation, oxidation and nitration adducts are consequently formed. Quantitative screening of these adducts in CSF may provide a biochemical indicator for the diagnosis of AD. To assess this, we measured 11 glycation adducts, three oxidation adducts and a nitration adduct, determining both protein adduct residues and free adducts, in CSF samples of age-matched normal healthy subjects (n = 18) and subjects with Alzheimer's disease (n = 32). In CSF protein, the concentrations of 3-nitrotyrosine, N(epsilon)-carboxymethyl-lysine, 3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine residues were increased in subjects with Alzheimer's disease. In CSF ultrafiltrate, the concentrations of 3-nitrotyrosine, methylglyoxal-derived hydroimidazolone and glyoxal-derived hydroimidazolone free adducts were also increased. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-nitrotyrosine residue concentration (p < 0.05), and the negative correlation with fructosyl-lysine residues just failed to reach significance (p = 0.052). Multiple linear regression gave a regression model of the MMSE score on 3-nitrotyrosine, fructosyl-lysine and N(epsilon)-carboxyethyl-lysine residues with p-values of 0.021, 0.031 and 0.052, respectively. These findings indicate that protein glycation, oxidation and nitration adduct residues and free adducts were increased in the CSF of subjects with Alzheimer's disease. A combination of nitration and glycation adduct estimates of CSF may provide an indicator for the diagnosis of Alzheimer's disease.     

Effects of oxidative and nitrosative stress in brain on p53 proapoptotic protein in amnestic mild cognitive impairment and Alzheimer disease

Many studies reported that oxidative and nitrosative stress might be important for the pathogenesis of Alzheimer's disease (AD) beginning with arguably the earliest stage of AD, i.e., as mild cognitive impairment (MCI). p53 is a proapoptotic protein that plays an important role in neuronal death, a process involved in many neurodegenerative disorders. Moreover, p53 plays a key role in the oxidative stress-dependent apoptosis. We demonstrated previously that p53 levels in brain were significantly higher in MCI and AD IPL (inferior parietal lobule) compared to control brains. In addition, we showed that in AD IPL, but not in MCI, HNE, a lipid peroxidation product, was significantly bound to p53 protein. In this report, we studied by means of immunoprecipitation analysis, the levels of markers of protein oxidation, 3-nitrotyrosine (3-NT) and protein carbonyls, in p53 in a specific region of the cerebral cortex, namely the inferior parietal lobule, in MCI and AD compared to control brains. The focus of these studies was to measure the oxidation and nitration status of this important proapoptotic protein, consistent with the hypothesis that oxidative modification of p53 could be involved in the neuronal loss observed in neurodegenerative conditions.     

Profiling proteins related to amyloid deposited brain of Tg2576 mice

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that is characterized by the extracellular deposition of beta-amyloid and intracellular hyperphosphorylation of tau in the cortex and hippocampus of the brain. These characterizations are caused by abnormal expression, modification and deposition of certain proteins. Post-translational modifications of proteins including oxidation and nitration might be involved in the pathogenesis of AD. In this study, AD-related proteins were identified in the cortex of Tg2576 mice used as a model for studying AD. Tg2576 mice express high levels of the Swedish mutated form of human beta-amyloid precursor protein (APP) and generated high levels of beta-amyloid in the brains. Using Western blotting and two-dimensional electrophoresis, proteins with differences in expression, oxidation and nitration in the cortex of Tg2576 mice brains were compared to littermate mice brains used as a control. The proteins with different expression levels were identified using matrix-assisted laser desorption/ionization-time of flight and liquid chromatography-tandem mass spectrometry analyses. As a result, 12 proteins were identified among 37 different proteins using the PDQuest program. Furthermore, two proteins, laminin receptor and alpha-enolase, were more susceptible to oxidative modification in the brains of Tg2576 mice compared to those of littermates. Similarly, alpha-enolase, calpain 12, and Atp5b were more modified by nitration in brains of Tg2576 mice than those of littermates. Taken together, these proteins and their modifications may play an important role in the plaque deposition of Tg2576 mice brains.     

Vulnerability of synaptosomes from apoE knock-out mice to structural and oxidative modifications induced by A beta(1-40): implications for Alzheimer's disease

Apolipoprotein E (apoE) plays an important role in the response to central nervous system injury. The e4 allele of apoE and amyloid beta-peptide (Abeta) are associated with Alzheimer's disease (AD) and may be central to the pathogenesis of this disorder. Recent studies demonstrate evidence for neurodegeneration and increased lipid peroxidation in transgenic mice lacking apoE (KO). In the current study, synaptosomes were prepared from apoE KO mice to determine the role of apoE in synaptic membrane structure and to determine susceptibility to oxidative damage by Abeta(1-40). ApoE KO mice exhibited structural modifications to lipid and protein components of synaptosomal membranes as determined by electron paramagnetic resonance in conjunction with lipid- and protein- specific spin labels. Incubation with 5 microM Abeta(1-40) resulted in more severe oxidative modifications to proteins and lipids in apoE KO synaptosomes as measured by protein carbonyls, an index of protein oxidation, and TBARs and protein-bound 4-hydroxynonenal (HNE), markers of lipid oxidation. Together, these data support a role for apoE in the modulation of oxidative injury and in the maintenance of synaptic integrity and are discussed with reference to alterations in AD brain.     

Redox proteomics: identification of oxidatively modified proteins

Reactive oxygen and nitrogen species may cause various types of chemical modifications on specific proteins, Such modifications if irreversible are often associated with permanent loss of function and may lead to the elimination or to the accumulation of the damaged proteins. Reversible modifications, particularly at the cysteine residues, may have a dual role of protection from cysteine irreversible oxidation and modulation of protein function (redox regulation). Here we will review the techniques available for identifying proteins based on their redox state. In particular, we will focus on protein carbonylation, tyrosine nitration and thiol-disulfide chemistry of cysteines, with special emphasis on glutathionylation, because these are the fields where the tools of proteome analysis have been applied.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB,glutamine synthase,and ubiquitin carboxy-terminal hydrolase L-1

Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and beta-actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer's disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurodegenerative disorders without the limitations of the immunochemical detection method.     

Protein damage in diabetes and uremia--identifying hotspots of proteome damage where minimal modification is amplified to marked pathophysiological effect

Increased protein glycation, oxidation and nitration are found in diabetes and renal failure. Steady state levels of glycated, oxidized and nitrated proteins are generally low, yet often have significant physiological effects--particularly linked to development and progression of vascular complications, including often fatal cardiovascular disease. Identification of sites activated toward damaging modifications or 'hotspots' in functional domains within proteins appears key to assessing targets of functional impairment. Disease progression is likely linked to instances where change in low level of hotspot damage influences metabolic control or physiological function. Examples discussed are: type IV collagen modification leading to endothelial cell detachment and anoikis, mitochondrial protein modification leading to oxidative stress and apolipoprotein B100 modification in low density lipoprotein leading to vascular retention and atherosclerosis. The role of mathematical systems biology, bioinformatics and proteome dynamics in future investigations is discussed.     

4-Hydroxynonenal-Derived Advanced Lipid Peroxidation End Products Are Increased in Alzheimer's Disease

Abstract: Recent studies have demonstrated oxidative damage is one of the salient features of Alzheimer's disease (AD). In these studies, glycoxidation adduction to and direct oxidation of amino acid side chains have been demonstrated in the lesions and neurons of AD. To address whether lipid damage may also play an important pathogenic role, we raised rabbit antisera specific for the lysine-derived pyrrole adducts formed by lipid peroxidation-derived 4-hydroxynonenal (HNE). These antibodies were used in immunocytochemical evaluation of brain tissue from AD and age-matched control patients. HNE-pyrrole immunoreactivity not only was identified in about half of all neurofibrillary tangles, but was also evident in neurons lacking neurofibrillary tangles in the AD cases. In contrast, few senile plaques were labeled, and then only the dystrophic neurites were weakly stained, whereas the amyloid-β deposits were unlabeled. Age-matched controls showed only background HNE-pyrrole immunoreactivity in hippocampal or cortical neurons. In addition to providing further evidence that oxidative stress-related protein modification is a pervasive factor in AD, the known neurotoxicity of HNE suggests that lipid peroxidation may also play a role in the neuronal death in AD that underlies cognitive deficits.     

Nitration in neurodegeneration: deciphering the "Hows" "nYs"

Recent literature has ushered in a new awareness of the diverse post-translational events that can influence protein folding and function. Among these modifications, protein nitration is thought to play a critical role in the onset and progression of several neurodegenerative diseases. While previously considered a late-stage epiphenomenon, nitration of protein tyrosine residues appears to be an early event in the lesions of amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. The advent of highly specific biochemical and immunological detection methods reveals that nitration occurs in vivo with biological selectively and site specificity. In fact, nitration of only a single Tyr residue is often sufficient to induce profound changes in the activity of catalytic proteins and the three-dimensional conformation of structural proteins. Presumably, nitration modifies protein function by altering the hydrophobicity, hydrogen bonding, and electrostatic properties within the targeted protein. Most importantly, however, nitrative injury may represent a unifying mechanism that explains how genetic and environmental causes of neurological disease manifest a singular phenotype. In this review and synthesis, we first examine the pathways of protein nitration in biological systems and the factors that influence site-directed nitration. Subsequently, we turn our attention to the structural implications of site-specific nitration and how it affects the function of several neurodegeneration-related proteins. These proteins include Mn superoxide dismutase and neurofilament light subunit in amyotrophic lateral sclerosis, alpha-synuclein and tyrosine hydroxylase in Parkinson's disease, and tau in Alzheimer's disease.     

Lipid peroxidation triggers neurodegeneration: A redox proteomics view into the Alzheimer disease brain

Lipid peroxidation involves a cascade of reactions in which production of free radicals occurs selectively in the lipid components of cellular membranes. Polyunsaturated fatty acids easily undergo lipid peroxidation chain reactions, which, in turn, lead to the formation of highly reactive electrophilic aldehydes. Among these, the most abundant aldehydes are 4-hydroxy-2-nonenal (HNE) and malondialdehyde, while acrolein is the most reactive. Proteins are susceptible to posttranslational modifications caused by aldehydes binding covalently to specific amino acid residues, in a process called Michael adduction, and these types of protein adducts, if not efficiently removed, may be, and generally are, dangerous for cellular homeostasis. In the present review, we focused the discussion on the selective proteins that are identified, by redox proteomics, as selective targets of HNE modification during the progression and pathogenesis of Alzheimer disease (AD). By comparing results obtained at different stages of the AD, it may be possible to identify key biochemical pathways involved and ideally identify therapeutic targets to prevent, delay, or treat AD.     

Oxidized neprilysin in aging and Alzheimer's disease brains

Deposition of amyloid in the brain is important in the pathogenesis of Alzheimer's disease (AD), but it remains to be determined if deposition is due to increased production or decreased clearance of fibrillogenic forms of beta-amyloid (Abeta). Except for rare genetic forms of AD, there is little evidence for increased production of Abeta, but decreases in enzymes involved in the clearance of Abeta are increasingly being investigated. Neprilysin (NEP) is a major enzyme for degradation of Abeta and changes in amount or activity of NEP may play a role in Abeta deposition in AD. Since oxidative damage to proteins, including formation of adducts such as 4-hydroxynonenal (HNE), has been reported in AD, it was of interest to determine if NEP might be susceptible to oxidative modification. To address this question, monoclonal antibody immunoprecipitates of NEP were probed with polyclonal antibodies to NEP and HNE. The results showed decreased NEP in AD compared to normal controls. NEP in both AD and controls had HNE-modification and the ratio of oxidized to total NEP was greater in AD than in controls. These findings suggest that decreased NEP may contribute to Abeta deposition in AD and that age-related oxidative damage to NEP may play a role in age-related cerebral amyloidosis that is exacerbated in AD.     

Oxidatively Modified GST and MRP1 in Alzheimer’s Disease Brain: Implications for Accumulation of Reactive Lipid Peroxidation Products

Alzheimer disease (AD) is a neurodegenerative disorder characterized pathologically by intracellular inclusions including neurofibrillary tangles (NFT) and senile plaques. Several lines of evidence implicate oxidative stress with the progression of AD. 4-hydroxy-2-trans-nonenal (HNE), an aldehydic product of membrane lipid peroxidation, is increased in AD brain. The alpha class of glutathione S-transferase (GST) can detoxify HNE and plays an important role in cellular protection against oxidative stress. The export of the glutathione conjugate of HNE is required to fully potentiate the GST-mediated protection. The multidrug resistance protein-1 (MRP1) and GST proteins may act in synergy to confer cellular protection. In the present study, we studied oxidative modification of GST and MRP1 in AD brain by immunoprecipitation of GST and MRP1 proteins followed by Western blot analysis using anti-HNE antibody. The results suggested that HNE is covalently bound to GST and MRP1 proteins in excess in AD brain. Collectively, the data suggest that HNE may be an important mediator of oxidative stress-induced impairment of this detoxifying system and may thereby play a role in promoting neuronal cell death. The results from this study also imply that augmenting endogenous oxidative defense capacity through dietary or pharmacological intake of antioxidants may slow down the progression of neurodegenerative processes in AD.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor-intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L-1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme alpha-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.     

Site-specific nitration and oxidative dityrosine bridging of the tau protein by peroxynitrite: implications for Alzheimer's disease

Alzheimer's disease (AD) is a progressive amnestic disorder typified by the pathological misfolding and deposition of the microtubule-associated tau protein into neurofibrillary tangles (NFTs). While numerous post-translational modifications influence NFT formation, the molecular mechanisms responsible for tau aggregation remain enigmatic. Since nitrative and oxidative injury have previously been shown to play a mechanistic role in neurodegeneration, we examined whether these events influence tau aggregation. In this report, we characterize the effects of peroxynitrite (ONOO-)-mediated nitration and oxidation on tau polymerization in vitro. Treatment of tau with ONOO- results in 3-nitrotyrosine (3-NT) immunoreactivity and the formation of heat-stable, SDS-insoluble oligomers. Using ESI-MS and HPLC with fluorescent detection, we show that these higher-order aggregates contain 3,3'-dityrosine (3,3'-DT). Tyrosine (Tyr) residues are critical for ONOO(-)-mediated oligomerization, as tau proteins lacking all Tyr residues fail to generate oligomers upon ONOO- treatment. Further, tau nitration targets residues Y18, Y29, and to a lesser degree Y197 and Y394, and nitration at these sites inhibits in vitro polymerization. The inhibitory effect of nitration on tau polymerization is specific for the 3-NT modification, as pseudophosphorylation at these same Tyr residues does not inhibit tau assembly. Our results suggest that the nitrative and oxidative roles of ONOO- differentially affect tau polymerization and that ONOO(-)-mediated cross-linking could facilitate tau aggregation in AD.     

Hydralazine modifies Aβ fibril formation and prevents modification by lipids in vitro

Lipid oxidative damage and amyloid β (Aβ) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aβ misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aβ production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aβ misfolding and (2) Aβ protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aβ aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aβ, and Aβ was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aβ and also did not prevent Aβ aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aβ formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aβ misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

Toxicity of extracellular secreted alpha-synuclein: Its role in nitrosative stress and neurodegeneration

It has been demonstrated that both oligomerisation and accumulation of α-synuclein (ASN) are the key molecular processes involved in the pathophysiology of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and other synucleinopathies. Alterations of ASN expression and impairment of its degradation can lead to the formation of intracellular deposits of this protein, called Lewy bodies. Overexpressed or misfolded ASN could be secreted to the extracellular space. Today the prion-like transmission of ASN oligomers to neighbouring cells is believed to be responsible for protein modification and propagation of neurodegeneration in the brain. It was presented that oxidative/nitrosative stress may play a key role in ASN secretion and spread of ASN pathology. Moreover, ASN-evoked protein oxidation, nitration and nitrosylation lead to disturbances in synaptic transmission and cell death. The interaction of secreted ASN with other amyloidogenic proteins and its involvement in irreversible mitochondrial disturbances and oxidative stress were also described. A better understanding of the mechanisms of ASN secretion and dysfunction may help to explain the molecular mechanisms of neurodegeneration and may be the basis for the development of novel therapeutic strategies.     

Involvement of PI3K/PKG/ERK1/2 signaling pathways in cortical neurons to trigger protection by cotreatment of acetyl-L-carnitine and alpha-lipoic acid against HNE-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Oxidative stress has been shown to underlie neuropathological aspects of Alzheimer's disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of lipid peroxidation of unsaturated lipids. HNE-induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. GSH plays a key role in antioxidant defense, and HNE exposure causes an initial depletion of GSH that leads to gradual toxic accumulation of reactive oxygen species. In the current study, we investigated whether pretreatment of cortical neurons with acetyl-L-carnitine (ALCAR) and alpha-lipoic acid (LA) plays a protective role in cortical neuronal cells against HNE-mediated oxidative stress and neurotoxicity. Decreased cell survival of neurons treated with HNE correlated with increased protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (HNE) accumulation. Pretreatment of primary cortical neuronal cultures with ALCAR and LA significantly attenuated HNE-induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose-dependent manner. Additionally, pretreatment of ALCAR and LA also led to elevated cellular GSH and heat shock protein (HSP) levels compared to untreated control cells. We have also determined that pretreatment of neurons with ALCAR and LA leads to the activation of phosphoinositol-3 kinase (PI3K), PKG, and ERK1/2 pathways, which play essential roles in neuronal cell survival. Thus, this study demonstrates a cross talk among the PI3K, PKG, and ERK1/2 pathways in cortical neuronal cultures that contributes to ALCAR and LA-mediated prosurvival signaling mechanisms. This evidence supports the pharmacological potential of cotreatment of ALCAR and LA in the management of neurodegenerative disorders associated with HNE-induced oxidative stress and neurotoxicity, including AD.