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The increasingly competitive biopharmaceutical industry requires companies to focus on rapid and low-cost cell line development. Single-cell cloning (SCC) is a critical and high-value process for cell line development, and typically problematic because single cell proliferates slowly when cultivated at low cell densities. Conditioned media (CM) provide autocrine growth factors to facilitate single cell proliferation, thus improve SCC efficiency. However, conditioned media (CM) are not a feasible solution for industrial cell line development due to variation and cross-contamination concerns. Here, we have found an improvement in the SCC efficiency similar to CM when soy hydrolysate was supplemented in SCC media. Therefore, we concluded that hydrolysate can mimic the autocrine growth factor(s) effect to improve cloning efficiency observed in CM.

A deeper understanding of the biological events occurring when bioprocess parameters changed will be of great value in improving the monoclonal antibodies (mAbs) production. Design of experiment (DoE) was applied to investigate the effect of process parameters (pH, temperature shift and dissolve oxygen (DO)) on protein titer. The key metabolites connecting the critical process parameters (CPPs) with monoclonal antibody production were identified by different chemometrics tools. Finally, the biological events of marker metabolites relating with titer improvement were concluded. pH and temperature shift were identified as CPPs that affect the target protein titer. A series of metabolites influenced by the altered CPPs and correlated with protein titer were screened by principal component analysis (PCA) and Pearson’ correlation test. The marker metabolites and their pathways linking CPPs to target protein titer in different culture phases were summarized. Metabolomics and chemometrics are promising data-driven tools to shine light into the biological black box between the bioprocess parameters and process performance.

Their ease of growth and transfection makes HEK293 cells a common cell culture in academic research. In addition, high transfection efficiency of HEK293 cells enable production of exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Recently, HEK293 cells has gained attention due to it is versatility for transfection experiments, particularly the propagation of adenoviral-based and retroviral-based vectors during CART-T bioprocess. Since traceability is critical to pharmaceutical manufacturing process, we provide a mini review to clarify the historical development and intent use of different variants of HEK293 cells. This review should provide a key reference for the HEK293 variants’ historical and developmental background.

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.

To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples.A statistical significant correlation between the sialic acid content and size-exclusion chromatography (SEC)-HPLC retention time of an Fc-fusion protein was observed when analyzing the titer of the samples. Using linear fitting analysis, the data fit the model well with R (2) = 0.985. Based on the SDS-PAGE and oligosaccharide analysis, we speculate that the amounts of the glycans could expand the structure of the Fc-fusion protein. This was manifested by the SEC-HPLC method in which proteins were separated based on its molecular size. In order to development a robust PAT method, an internal standard was used to improve the precision of the method by reducing systematic errors. We found the change of SEC retention time (delta t) and sialic acid content were highly correlated (R (2) = 0.992). This method was further validated by a 1500 l production process.SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.

The formation of "Advanced Glycation End products" (AGEs) is an inevitable consequence of mammalian glucose metabolism. AGE-mediated protein-protein crosslinks lead to detergent-insoluble and protease-resistant protein aggregates, and in Alzheimer's disease (AD) extra cellular senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs) have been shown to contain AGEs. However, to date little is known concerning the most prevalent protein-targets of AGE modification under normal, non-pathological conditions. Here, a combination of 2D-electrophoresis, Western blotting and mass spectrometry has been used to identify preferentially AGE-modified proteins in oligodendrocyte (OLN-93) and neuroblastoma cell lines (SH-SY5Y) in standard culture. Proteomics analysis identified a total of eight targets with structural, metabolic and regulatory function, three of which (beta-actin, beta-tubulin and eukaryotic Elongation Factor 1-alpha) were common to both cell lines. Based on results from prior studies, modification of these proteins may lead to a loss of function. Consequently, the identification of targets for these proteins is of particular interest for a better understanding of the consequences of AGE-modification in aging, neurodegenerative diseases and diabetes.

The brain is susceptible to oxidative stress, which is associated with age-related brain dysfunction, because of its high content of peroxidizable unsaturated fatty acids, high oxygen consumption per unit weight, high content of key components for oxidative damage, and the relative scarcity of antioxidant defense systems. Protein oxidation, which results in functional disruption, is not random but appears to be associated with increased oxidation in specific proteins. By using a proteomics approach, we have compared the protein levels and specific protein carbonyl levels, an index of oxidative damage in the brains of old mice, to these parameters in the brains of young mice and have identified specific proteins that are altered as a function of aging. We show here that the expression levels of dihydropyrimidinase-like 2 (DRP2), alpha-enolase (ENO1), dynamin-1 (DNM1), and lactate dehydrogenase 2 (LDH2) were significantly increased in the brains of old versus young mice; the expression levels of three unidentified proteins were significantly decreased. The specific carbonyl levels of beta-actin (ACTB), glutamine synthase (GS), and neurofilament 66 (NF-66) as well as a novel protein were significantly increased, indicating protein oxidation, in the brains of old versus young mice. These results were validated by immunochemistry. In addition, enzyme activity assays demonstrated that oxidation was associated with decreased GS activity, while the activity of lactate dehydrogenase was unchanged in spite of an up-regulation of LDH2 levels. Several of the up-regulated and oxidized proteins in the brains of old mice identified in this report are known to be oxidized in neurodegenerative diseases as well, suggesting that these proteins may be particularly susceptible to processes associated with neurodegeneration. Our results establish an initial basis for understanding protein alterations that may lead to age-related cellular dysfunction in the brain.

Alzheimer disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFT) and of extracellular senile plaques (SP), the central core of which is amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP), a transmembrane protein. AD brain has been reported to be under oxidative stress that may play an important role in the pathogenesis and progression of AD. The present proteomics study is focused on identification of a specific target of protein oxidation in AD hippocampus that has relevance to the role of oxidative stress in AD. Here, we report that the protein, Pin1, is significantly down-regulated and oxidized in AD hippocampus. The identity of Pin1 was confirmed immunochemically. Analysis of Pin1 activity in AD brain and separately as oxidized pure Pin1 demonstrated that oxidation of Pin1 led to loss of activity. Pin1 has been implicated in multiple aspects of cell cycle regulation and dephosphorylation of tau protein as well as in AD. The in vivo oxidative modification of Pin1 as found by proteomics in AD hippocampus in the present study suggests that oxidative modification may be related to the known loss of Pin1 isomerase activity that could be crucial in AD neurofibrillary pathology. Taken together, these results provide evidence supporting a direct link between oxidative damage to neuronal Pin1 and the pathobiology of AD.

Age-related impairment of functionality of the central nervous system (CNS) is associated with increased susceptibility to develop many neurodegenerative diseases. Increased oxidative stress in the CNS of aged animals is manifested by increased protein oxidation, which is believed to contribute to the age-related learning and memory deficits. Glutamate dysregulation, mitochondrial dysfunction and impaired protein synthesis are observed in aged brains, along with increased protein oxidation. Interestingly, all of these age-related cellular alterations can be improved by caloric restriction (CR), which can also improve the plasticity and recovery of the CNS. Although the beneficial effects of CR on brains are well established, the mechanism(s) of its action remains unclear. In order to gain insight into the mechanism of CR in the brain, we located the brain regions that are benefited the most from reduced oxidative stress by CR. Along with other brain regions, striatum (ST) showed significantly decreased bulk protein carbonyl levels and hippocampus (HP) showed decreased bulk protein 3-nitrotyrosine (3-NT) levels in CR aged rats when compared to those of age matched controls. To determine which proteins were oxidatively modified in these brain regions, we used parallel proteomics approach to identify the proteins that are altered in oxidation and expression. The specific carbonyl levels of pyruvate kinase M2 (PKM2), alpha-enolase (ENO1), inositol monophosphatase (INSP1), and F1-ATPase Chain B (ATP-F1B) were significantly decreased in ST of aged CR rats. In contrast, the expression levels of phosphoglycerate kinase 1 (PKG1), inosine monophosphate cyclohydrolase (IMPCH) and F1-ATPase Chain A (ATP-F1A) were significantly increased in the ST of CR rats. In the hippocampus of CR rats, the specific 3-NT levels of malate dehydrogenase (MDH), phosphoglycerate kinase 1 (PKG1) and 14-3-3 zeta protein were significantly decreased and expression levels of DLP1 splice variant 1 (DLP1), mitochondrial aconitase (ACO2), dihydrolipoamide dehydrogenase (DLDH), neuroprotective peptide H3 (NPH3), and eukaryotic translation initiation factor 5A (eIF-5A) are increased. Moreover, an unnamed protein product (UNP1) with similar sequence to initiation factor 2 (IF-2) was decreased in the HP of CR rats. Our data support the hypothesis that CR induces a mild metabolic stress response by increasing the production of neurotrophic proteins, therefore, priming neurons against apoptosis. Moreover, our study shows that the improvement of glutamate dysregulation, mitochondrial dysfunction and protein synthesis by CR is, at least partially, due to the CR-mediated alteration of the oxidation or the expression of PKM2, ENO1, INSP1, ATP-F1B, PKG1, IMPCH, ATP-F1A MDH, PKG1 and 14-3-3 zeta protein, DLP1, ACO2, DLDH, NPH3, eIF-5A and UNP1. This study provides valuable insights into the mechanisms of the beneficial factors on brain aging by CR.

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

The senescence-accelerated mouse (SAM) is an accelerated aging model that was established through phenotypic selection from a common genetic pool of AKR/J strain of mice. The SAM model was established in 1981, including nine major senescence-accelerated mouse prone (SAMP) substrains and three major senescence-accelerated mouse resistant (SAMR) substrains, each of which exhibits characteristic disorders. Recently, SAMP8 have drawn attention in gerontological research due to its characteristic learning and memory deficits at old age. Many recent reports provide insight into mechanisms of the cognitive impairment and pathological changes in SAMP8. Therefore, this mini review examines the recent findings of SAMP8 mice abnormalities at the gene and protein levels. The genes and proteins described in this review are functionally categorized into neuroprotection, signal transduction, protein folding/degradation, cytoskeleton/transport, immune response and reactive oxygen species (ROS) production. All of these processes are involved in learning and memory. Although these studies provide insight into the mechanisms that contribute to the learning and memory decline in aged SAMP8 mice, higher throughput techniques of proteomics and genomics are necessary to study the alterations of gene expression and protein abnormalities in SAMP8 mice brain in order to more completely understand the central nervous system dysfunction in this mouse model. The SAMP8 is a good animal model to investigate the fundamental mechanisms of age-related learning and memory deficits at the gene and protein levels.

Amyloid beta-peptide (Abeta) is the major constituent of senile plaques, a pathological hallmark of Alzheimer's disease (AD) brain. It is generally accepted that Abeta plays a central role in the pathophysiology of AD. Abeta is released from cells under entirely normal cellular conditions during the internalization and endosomal processing of amyloid precursor protein (APP). However, accumulation of Abeta can induce neurotoxicity. Our previous reports showed that decreasing the production of Abeta by giving an intracerebroventricular injection of a 42-mer phosphorothiolated antisense oligonucleotide (AO) directed at the Abeta region of the APP gene reduces lipid peroxidation and protein oxidation and improves cognitive deficits in aged senescence-accelerated mice prone 8 (SAMP8) mice. In order to investigate how Abeta level reduction improves learning and memory performance of SAMP8 mice through reduction of oxidative stress in brains, we used proteomics to identify the proteins that are less oxidized in 12-month-old SAMP8 mice brains treated with AO against the Abeta region of APP (12 mA) compared to that of the age-control SAMP8 mice. We found that the specific protein carbonyl levels of aldoase 3 (Aldo3), coronin 1a (Coro1a) and peroxiredoxin 2 (Prdx2) are significantly decreased in the brains of 12 mA SAMP8 mice compared to the age-controlled SAMP8 treated with random AO (12 mR). We also found that the expression level of alpha-ATP synthase (Atp5a1) was significantly decreased, whereas the expression of profilin 2 (Pro-2) was significantly increased in brains from 12 mA SAMP8 mice. Our results suggest that decreasing Abeta levels in aged brain in aged accelerated mice may contribute to the mechanism of restoring the learning and memory improvement in aged SAMP8 mice and may provide insight into the role of Abeta in the memory and cognitive deficits in AD.

Decline in olfactory ability has been associated with aging as well as neurodegenerative disorders. The aim of this study was to gain fundamental insight into molecular events associated with the aging olfactory system. We report a comparative proteomic analysis of the olfactory epithelium (OE) and olfactory bulb (OB) of old (80-week old) and young (6-week old) mice with further analysis of age-related differences in differentially expressed proteins at the mRNA level using real-time RT-PCR. Nine proteins in the OE and 20 in the OB were differentially expressed in old and young mice; of these, aldolase 1, peptidyl prolyl isomerase A, mitochondrial aconitase 2, mitochondrial aldehyde dehydrogenase 2 and albumin 1 were identified in the OE; and ATP synthase isoform 1, enolase 1, ferritin heavy chain, malate dehydrogenase 1, tropomyosin alpha 3 chain and dynamin 1 were identified in the OB. At the transcriptional level, aconitase 2 in the OE and ferritin heavy chain 1 in the OB were differentially expressed with aging, in concordance with the proteomic data. Our results demonstrate an altered proteomic profile of the aged murine olfactory system. The identified proteins fall into three broadly defined functional categories: (i) metabolism, (ii) transport/motility and (iii) stress response. Our transcriptional analysis provides insight into possible mechanisms by which protein expression may be regulated in the OE and OB. The results are discussed in relation to the decrement in olfactory sensitivity with aging.

Protein oxidation has been implicated in Alzheimer's disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, alpha-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1-42 (Abeta 1-42) leads to the formation of protein carbonyls. In order to test the involvement of Abeta(1-42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Abeta(1-42) treatment leads to oxidatively modified proteins, consistent with the notion that Abeta(1-42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified beta-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Abeta(1-42). Additionally, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, gamma-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD.

Astrocyte infection in HIV has been associated with rapid progression of dementia in a subset of HIV/AIDS patients. Astrogliosis and microglial activation are observed in areas of axonal and dendritic damage in HIVD. In HIV-infected astrocytes, the regulatory gene tat is over expressed and mRNA levels for Tat are elevated in brain extracts from individuals with HIV-1 dementia. Tat can be detected in HIV-infected astrocytes in vivo. The HIV-1 protein Tat transactivates viral and cellular gene expression, is actively secreted mainly from astrocytes, microglia and macrophages, into the extracellular environment, and is taken up by neighboring uninfected cells such as neurons. The HIV-1 protein Tat released from astrocytes reportedly produces trimming of neurites, mitochondrial dysfunction and cell death in neurons, while protecting its host, the astrocyte. We utilized proteomics to investigate protein expression changes in human astrocytes intracellularly expressing Tat (SVGA-Tat). By coupling 2D fingerprinting and identification of proteins by mass spectrometry, we identified phosphatase 2A, isocitrate dehydrogenase, nuclear ribonucleoprotein A1, Rho GDP dissociation inhibitor alpha, beta-tubulin, crocalbin like protein/calumenin, and vimentin/alpha-tubulin to have decreased protein expression levels in SVGA-Tat cells compared to the SVGA-pcDNA cells. Heat shock protein 70, heme oxygenase-1, and inducible nitric oxide synthase were found to have increased protein expression in SVGA-Tat cells compared to controls by slotblot technique. These findings are discussed with reference to astrocytes serving as a reservoir for the HIV virus and how Tat promotes survival of the astrocytic host.

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.

Amyloid beta-peptide (Abeta) is known to induce free radical-mediated oxidative stress in the brain. Free radical-mediated damage to the neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD). Abeta is produced by proteolytic processing of the amyloid precursor protein (APP). The senescence accelerated mouse prone 8 (SAMP8) strain was developed by phenotypic selection from a common genetic pool. The SAMP8 strain exhibits age-related deterioration in memory and learning as well as Abeta accumulation, and it is considered an effective model for studying brain aging in accelerated senescence. Previous research has shown that a phosphorothiolated antisense oligonucleotide directed against the Abeta region of APP decreases the expression of APP and reverses deficits in learning and memory in aged SAMP8 mice. Consistent with other reports, our previous study showed that 12-month-old SAMP8 mice have increased levels of oxidative stress markers in the brain compared with that in brains from 4-month-old SAMP8 mice. In the current study, 12-month-old SAMP8 mice were treated with antisense oligonucleotide directed against the Abeta region of APP, and the oxidative markers in brain were decreased significantly. Therefore, we conclude that Abeta may contribute to the oxidative stress found in aged SAMP8 mice that have learning and memory impairments. These results are discussed in reference to AD.

Aging is one of the unique features in all organisms. The impaired functional capacity of many systems characterizes aging. When such impairments occur in the brain, the susceptibility to neurodegenerative diseases amplifies considerably. The free radical theory of aging posits that the functional impairments in brains are due to the attack on critical cellular components by free radicals, reactive oxygen species, and reactive nitrogen species produced during normal metabolism. In this review, we examine this concept based on the parameters of oxidative stress in correlation to aging. The parameters for lipid peroxidation are phospholipid composition, reactive aldehydes, and isoprostanes. The parameters for protein oxidation are protein carbonyl levels, protein 3-nitrotyrosine levels, electron paramagnetic resonance, and oxidative stress-sensitive enzyme activities. We conclude that free radicals are, at least partially, responsible for the functional impairment in aged brains. The aging brain, under oxidative stress, responds by induction of various protective genes, among which is heme oxygenase. The products of the reaction catalyzed by heme oxygenase, carbon monoxide, iron, and biliverdin (later to bilirubin) each have profound effects on neurons. Although there may be other factors contributing to brain aging, free radicals are involved in the damaging processes associated with brain aging, and cellular stress response genes are induced under free radical oxidative stress. Therefore, this review supports the proposition that free radicals are, indeed, a key to brain aging.

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, a-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Ab and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood‐brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence

Oxidative stress, an imbalance between the oxidant and antioxidant systems, has been implicated in the pathogenesis of numerous neurodegenerative diseases [1]. Among all the body organs, the brain is particularly vulnerable to oxidative damage because of its high utilization of oxygen, increased levels of polyunsaturated fatty acids, and relatively high levels of redox transition metal ions in certain brain regions; in addition, the brain has relatively low levels of antioxidants [2–6]. The presence of iron ion in an oxygen-rich environment can further lead to enhanced production of superoxide radicals and ultimately to a cascade of oxidative events. Either the oxidant directly or the products of oxidative stress could trigger the oxidative modification of a number of cellular macromolecular targets, including proteins, lipids, DNA, RNA, and carbohydrates, which may lead to impairment of cellular functions [2,3,5,7–9]. Among the earliest of these changes following an oxidative insult are increased levels of toxic carbonyls, 3-nitrotyrosine (3-NT), and 4-hydroxy-2trans-nonenal (HNE) [2,4,7,10–13]. HNE is derived from free radical attack on unsaturated acyl chains of phospholipids, particularly arachidonic acid. Oxidation leads to introduction of carbonyl groups to proteins [14]. Carbonyl groups are incorporated into proteins by direct oxidation of certain amino acid side chains, peptide backbone scission, or Michael addition reactions with products of lipid peroxidation or glycol oxidation [4,15,16]. Protein carbonyls can be detected by the derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), followed by immunochemical detection of the hydrazone product [14]. Oxidative stress can stimulate additional damage via overexpression of inducible nitric oxide synthase (iNOS) and the action of constitutive

Objective: To identify oxidatively modified proteins in brains of persons with inherited Alzheimer's disease. Methods: Redox proteomics was used to identify oxidatively modified brain proteins in persons with mutations in the genes for presenilin-1 (PS-1). Results: An initial redox proteomics assessment of oxidatively modified proteins from brains of individuals with PS-1 mutations was performed. These PS1 mutations, Q222H and M233T, are completely penetrant causing early-onset familial AD as previously reported in these Australian families. We show that oxidative modifications of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), γ-enolase, actin, and dimethylarginine dimethylaminohydrolase 1 (DMDMAH-1) are present in the brain of familial AD subjects. Conclusions: These initial results suggest that oxidatively modified proteins are important common features in both familial and sporadic AD.

A recent proteomics analysis from our laboratory demonstrated that several oxidative stress response proteins showed significant changes in steady-state levels in olfactory bulbs (OBs) of 20- vs. 1.5-month-old mice. Oxidative stress may result in protein oxidation. In this study, we investigated two forms of protein oxidative modification in murine OBs: carbonylation and nitration. Redox proteomics with two-dimensional gel electrophoresis, Western blotting, protein digestion, and mass spectrometry was used to quantify total and specific protein carbonylation and to identify differentially carbonylated proteins and determine the carbonylation status of previously identified proteins in OBs of 1.5- and 20-month-old mice. Immunohistochemistry was used to demonstrate the relative intensity and localization of protein nitration in OBs of 1.5-, 6-, and 20-month-old mice. Total protein carbonylation was significantly greater in OBs of 20- vs. 1.5-month-old mice. Aldolase 1 (ALDO1) showed significantly more carbonylation in OBs from 20- vs. 1.5-month-old mice; heat shock protein 9A and dihydropyrimidinase-like 2 showed significantly less. Several previously investigated proteins were also carbonylated, including ferritin heavy chain (FTH). Nitration, identified by 3-nitrotyrosine immunoreactivity, was least abundant at 1.5 months, intermediate at 6 months, and greatest at 20 months and was localized primarily in blood vessels. Proteins that were specific targets of oxidation were also localized: ALDO1 in astrocytes of the granule cell layer and FTH in mitral/tufted cells. These results indicate that specific carbonylated proteins, including those in astrocytes and mitral/tufted neurons, and nitrated proteins in the vasculature are molecular substrates of age-related olfactory dysfunction.

By 2003, an estimated 34 million Americans had used cocaine according to the National Survey on Drug Use & Health. About 5.9 million of those had used in the past 12 months. Chronic cocaine users often develop addiction, dependency and tolerance to the drug. The psychological and physical effects of cocaine are due to the disruption of the limbic system in the central nervous system (CNS). Increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the CNS. Although it is evident that cocaine induces oxidative stress in the CNS, little has been learned about whether such increased oxidative stress is also relevant to apoptosis in cocaine-exposed models. To gain insight into the role of cocaine-induced oxidative stress in apoptosis, we hypothesized that oxidative stress precedes cell death when cocaine is administrated. To test this hypothesis, we have monitored the oxidative stress and apoptotic effects of acute cocaine exposure in human neuronal progenitor cells (HNPC). We found that oxidative stress was significantly increased at 48 h after a 30 min cocaine exposure compared to control cells, and that this was followed by cell death at 72 h. Using the same experimental paradigm we have previously shown that pro-inflammatory genes are up-regulated in cocaine-exposed HNPC at 24 h. Therefore, we suggest that the increased oxidative stress (possibly mediated by inflammatory responses) precedes cell death in cocaine-exposed HNPC. This may have implications for the consequences of cocaine abuse in situations where antioxidant capacity is compromised, as in the aging brain.

Impaired function of the central nervous system (CNS) in aged animals is associated with increased susceptibility to the development of many neurodegenerative diseases. Age-related functional deterioration in brain is consistent with the free radical theory of aging that predicts, among other things, that free radical reactions with and damage to biomolecules, such as proteins and membrane lipid bilayers, leads to loss of neurons and subsequently diminished cognition. These oxidatively modified biomolecules are believed to contribute to the decreased antioxidant content, mitochondrial dysfunction, and impaired plasticity in aged brains. Treatment of rodents with L-acetylcarnitine (LAC; gamma-trimethyl-beta-acetylbutyrobetaine) can improve these functional losses. Although it is well established that administration of LAC can decrease protein oxidation in aged brains, it is not clear which proteins are decreased in their level of oxidation in the brains of aged rats treated with LAC. The current study used a parallel redox proteomics approach to identify the proteins that are oxidized in aged rat cortex and hippocampus of aged rats. Moreover, those proteins that are reduced in oxidation status were identified in aged brains from rats treated in vivo with LAC. The findings are discussed in reference to brain aging and age-related cognitive impairment.

Mild cognitive impairment (MCI) is generally referred to the transitional zone between normal cognitive function and early dementia or clinically probable Alzheimer's disease (AD). Oxidative stress plays a significant role in AD and is increased in the superior/middle temporal gyri of MCI subjects. Because AD involves hippocampal-resident memory dysfunction, we determined protein oxidation and identified the oxidized proteins in the hippocampi of MCI subjects. We found that protein oxidation is significantly increased in the hippocampi of MCI subjects when compared to age- and sex-matched controls. By using redox proteomics, we determined the oxidatively modified proteins in MCI hippocampus to be alpha-enolase (ENO1), glutamine synthetase (GLUL), pyruvate kinase M2 (PKM2) and peptidyl-prolyl cis/trans isomerase 1 (PIN1). The interacteome of these proteins revealed that these proteins functionally interact with SRC, hypoxia-inducible factor 1, plasminogen (PLG), MYC, tissue plasminogen activator (PLAT) and BCL2L1. Moreover, the interacteome indicates the functional involvement of energy metabolism, synaptic plasticity and mitogenesis/proliferation. Therefore, oxidative inactivation of ENO1, GLUL and PIN1 may alter these cellular processes and lead to the development of AD from MCI. We conclude that protein oxidation plays a significant role in the development of AD from MCI and that the oxidative inactivation of ENO1, GLUL, PKM2 and PIN1 is involved in the progression of AD from MCI. The current study provides a framework for future studies on the development of AD from MCI relevant to oxidative stress.

Decline in olfactory ability has been associated with aging as well as neurodegenerative disorders. The aim of this study was to gain fundamental insight into molecular events associated with the aging olfactory system. We report a comparative proteomic analysis of the olfactory epithelium (OE) and olfactory bulb (OB) of old (80-week old) and young (6-week old) mice with further analysis of age-related differences in differentially expressed proteins at the mRNA level using real-time RT-PCR. Nine proteins in the OE and 20 in the OB were differentially expressed in old and young mice; of these, aldolase 1, peptidyl prolyl isomerase A, mitochondrial aconitase 2, mitochondrial aldehyde dehydrogenase 2 and albumin 1 were identified in the OE; and ATP synthase isoform 1, enolase 1, ferritin heavy chain, malate dehydrogenase 1, tropomyosin alpha 3 chain and dynamin 1 were identified in the OB. At the transcriptional level, aconitase 2 in the OE and ferritin heavy chain 1 in the OB were differentially expressed with aging, in concordance with the proteomic data. Our results demonstrate an altered proteomic profile of the aged murine olfactory system. The identified proteins fall into three broadly defined functional categories: (i) metabolism, (ii) transport/motility and (iii) stress response. Our transcriptional analysis provides insight into possible mechanisms by which protein expression may be regulated in the OE and OB. The results are discussed in relation to the decrement in olfactory sensitivity with aging.

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles, senile plaques and loss of synapses. There is accumulating evidence that oxidative stress plays an important role in AD pathophysiology. Previous redox proteomics studies from our laboratory on AD inferior parietal lobule led to the identification of oxidatively modified proteins that were consistent with biochemical or pathological alterations in AD. The present study was focused on the identification of specific targets of protein oxidation in AD and control hippocampus and cerebellum using a redox proteomics approach. In AD hippocampus, peptidyl prolyl cis-trans isomerase, phosphoglycerate mutase 1, ubiquitin carboxyl terminal hydrolase 1, dihydropyrimidinase related protein-2 (DRP-2), carbonic anhydrase II, triose phosphate isomerase, alpha-enolase, and gamma-SNAP were identified as significantly oxidized protein with reduced enzyme activities relative to control hippocampus. In addition, no significant excessively oxidized protein spots were identified in cerebellum compared to control, consistent with the lack of pathology in this brain region in AD. The identification of oxidatively modified proteins in AD hippocampus was verified by immunochemical means. The identification of common oxidized proteins in different brain regions of AD brain suggests a potential role for these oxidized proteins and thereby oxidative stress in the pathogenesis of Alzheimer's disease.

Protein oxidation mediated by amyloid beta-peptide (1-42) (Abeta[1-42]) has been proposed to play a central role in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder associated with aging and the loss of cognitive function. The specific mechanism by which Abeta(1-42), the primary component of the senile plaque and a pathologic hallmark of AD, contributes to the oxidative damage evident in AD brain is unknown. Moreover, the specific proteins that are vulnerable to oxidative damage induced by Abeta(1-42) are unknown. Identification of such proteins could contribute to our understanding of not only the role of Abeta(1-42) in the pathogenesis of AD, but also provide insight into the mechanisms of neurodegeneration at the protein level in AD. We report the proteomic identification of two proteins found to be oxidized significantly in neuronal cultures treated with Abeta(1-42): 14-3-3zeta and glyceraldehyde-3-phosphate dehydrogenase. We also report that pretreatment of neuronal cultures with gamma-glutamylcysteine ethyl ester, a compound that supplies the limiting substrate for the synthesis of glutathione and results in the upregulation of glutathione in neuronal cultures, protects both proteins against Abeta(1-42)-mediated protein oxidation.

Amyotrophic lateral sclerosis (ALS) is an age-related, fatal motor neuron degenerative disease occurring both sporadically (sALS) and heritably (fALS), with inherited cases accounting for approximately 10% of diagnoses. Although multiple mechanisms likely contribute to the pathogenesis of motor neuron injury in ALS, recent advances suggest that oxidative stress may play a significant role in the amplification, and possibly the initiation, of the disease. Lipid peroxidation is one of the several outcomes of oxidative stress. Since the central nervous system (CNS) is enriched with polyunsaturated fatty acids, it is particularly vulnerable to membrane-associated oxidative stress. Peroxidation of cellular membrane lipids or circulating lipoprotein molecules generates highly reactive aldehydes, among which is 4-hydroxy-2-nonenal (HNE). HNE levels are increased in spinal cord motor neurons of ALS patients, indicating that lipid peroxidation is associated with the motor neuron degeneration in ALS. In the present study, we used a parallel proteomic approach to identify HNE-modified proteins in the spinal cord tissue of a model of fALS, G93A-SOD1 transgenic mice, in comparison to the nontransgenic mice. We found three significantly HNE-modified proteins in the spinal cord of G93A-SOD1 transgenic mice: dihydropyrimidinase-related protein 2 (DRP-2), heat-shock protein 70 (Hsp70), and possibly alpha-enolase. These results support the role of oxidative stress as a major mechanism in the pathogenesis of ALS. Structural alteration and activity decline of functional proteins may consistently contribute to the neurodegeneration process in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease characterized by the loss of neuronal function in the motor cortex, brain stem, and spinal cord. Familial ALS cases, accounting for 10-15% of all ALS disease, are caused by a gain-of-function mutation in Cu,Zn-superoxide dismutase (SOD1). Two hypotheses have been proposed to explain the toxic gain of function of mutant SOD (mSOD). One is that mSOD can directly promote reactive oxygen species and reactive nitrogen species generation, whereas the other hypothesis suggests that mSODs are prone to aggregation due to instability or association with other proteins. However, the hypotheses of oxidative stress and protein aggregation are not mutually exclusive. G93A-SOD1 transgenic mice show significantly increased protein carbonyl levels in their spinal cord from 2 to 4 months and eventually develop ALS-like motor neuron disease and die within 5-6 months. Here, we used a parallel proteomics approach to investigate the effect of the G93A-SOD1 mutation on protein oxidation in the spinal cord of G93A-SOD1 transgenic mice. Four proteins in the spinal cord of G93A-SOD1 transgenic mice have higher specific carbonyl levels compared to those of non-transgenic mice. These proteins are SOD1, translationally controlled tumor protein (TCTP), ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1), and, possibly, alphaB-crystallin. Because oxidative modification can lead to structural alteration and activity decline, our current study suggests that oxidative modification of UCH-L1, TCTP, SOD1, and possibly alphaB-crystallin may play an important role in the neurodegeneration of ALS.

Protein oxidation has been shown to lead to loss of protein function, increased protein aggregation, decreased protein turnover, decreased membrane fluidity, altered cellular redox poteintial, loss of Ca2+ homeostaisis, and cell death. There is increasing evidence that protein oxidation is involved in the pathogenesis of Alzheimer's disease and amyloid beta-peptide (1-42) has been implicated as a mediator of oxidative stress in AD. However, the specific implications of the oxidation induced by Abeta(1-42) on the neurodegeneration evident in AD are unknown. In this study, we used proteomic techniques to identify specific targets of oxidation in transgenic Caenorhabditis elegans (C. elegans) expressing human Abeta(1-42). We identified 16 oxidized proteins involved in energy metabolism, proteasome function, and scavenging of oxidants that are more oxidized compared to control lines. These results are discussed with reference to Alzheimer's disease.

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial function is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. Intracellularly expressed Tat is not toxic to astrocytes. In fact, intracellularly expressed Tat offers protection against oxidative stress-related toxins such as the mitochondrial toxin 3-nitroproprionic acid (3-NP). In the current study, human astrocytes expressing Tat (SVGA-Tat) and vector controls (SVGA-pcDNA) were each treated with the irreversible mitochondrial complex II inhibitor 3-NP. Proteomics analysis was utilized to identify changes in protein expression levels. By coupling 2D fingerprinting and identification of proteins by mass spectrometry, actin, heat shock protein 90, and mitochondrial single-stranded DNA binding protein were identified as proteins with increased expression, while lactate dehydrogenase had decreased protein expression levels in SVGA-Tat cells treated with 3-NP compared to SVGA-pcDNA cells treated with 3-NP. Oxidative damage can lead to several events including loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, ultimately leading to neuronal death. Identification of specific proteins protected from oxidation is a crucial step in understanding the interaction of Tat with astrocytes. In the current study, proteomics also was used to identify proteins that were specifically oxidized in SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. We found beta-actin, calreticulin precursor protein, and synovial sarcoma X breakpoint 5 isoform A to have increased oxidation in control SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. These results are discussed with reference to potential involvement of these proteins in HIV dementia and protection of astrocytes against oxidative stress by the HIV virus, a prerequisite for survival of a viral host cell.

We reviewed here the formation of free radicals and its effect physiologically. Studies mentioned above have indicated that free radical/ROS/RNS involvement in brain aging is direct as well as correlative. Increasing evidence demonstrates that accumulation of oxidation of DNA, proteins, and lipids by free radicals are responsible for the functional decline in aged brains. Also, lipid peroxidation products, such as MDA, HNE, and acrolein, were reported to react with DNA and proteins to produce further damage in aged brains. Therefore, the impact of free radicals on brain aging is pronounced. It has been estimated that 10,000 oxidative interactions occur between DNA and endogenously generated free radicals per human cell per day, and at least one of every three proteins in the cell of older animals is dysfunctional as an enzyme or structural protein, due to oxidative modification. Although these estimated numbers reveal that free radical-mediated protein and DNA modification play significant roles in the deterioration of aging brain, they do not imply that free radical damages are the only cause of functional decline in aged brain. Nevertheless,although other factors may be involved in the cascade of damaging effects in the brain, the key role of free radicals in this process cannot be underestimated. This article has examined the role and formation of free radicals in brain aging. We propose that free radicals are critical to cell damage in aged brain and endogenous, and that exogenous antioxidants, therefore, may play effective roles in therapeutic strategies for age-related neurodegenerative disorders.

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.