Biomedical subjects

D Allan Butterfield

Publications and source records attributed to D Allan Butterfield.

At least 55 records · Page 3Linked to original sources

PubMed2005

Lentivirus infection causes neuroinflammation and neuronal injury in dorsal root ganglia: pathogenic effects of STAT-1 and inducible nitric oxide synthase.

Distal sensory polyneuropathy (DSP) is currently the most common neurological complication of HIV infection in the developed world and is characterized by sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. We previously showed that feline immunodeficiency virus (FIV) infection of cats caused DSP together with immunosuppression in cats, similar to that observed in HIV-infected humans. In this study, we investigated the pathogenic mechanisms underlying the development of FIV-induced DSP using feline dorsal root ganglia (DRG) cultures, consisting of neurons, Schwann cells, and macrophages. FIV-infected cultures exhibited viral Ags (p24 and envelope) in macrophages accompanied by neuronal injury, indicated by neurite retraction, neuronal loss and decreased soma size, compared with mock-infected (control) cultures. FIV infection up-regulated inducible NO synthase (iNOS), STAT-1, and TNF-alpha mRNA levels in DRG cultures. Increased STAT-1 and iNOS mRNA levels were also observed in DRGs from FIV-infected animals relative to mock-infected controls. Similarly, immunolabeling studies of DRGs from FIV-infected animals showed that macrophages were the principal sources of STAT-1 and iNOS protein production. The iNOS inhibitor aminoguanidine reduced nitrotyrosine and protein carbonyl levels, together with preventing neuronal injury in FIV-infected DRG cultures. The present studies indicate that FIV infection of DRGs directly contributes to axonal and neuronal injury through a mechanism involving macrophage immune activation, which is mediated by STAT-1 and iNOS activation.

Animals↗

PubMed2005

Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: Mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis.

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.

Aging↗

PubMed2005

Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.

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.

Actins↗

PubMed2005

Proteomic analysis of protein expression and oxidative modification in r6/2 transgenic mice: a model of Huntington disease.

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.

Aging↗

PubMed2005

Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich's ataxia.

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) associated with mitochondrial dysfunction. The mitochondrial genome may play an essential role in the pathogenesis of these diseases, and evidence for mitochondria being a site of damage in neurodegenerative disorders is based in part on observed decreases in the respiratory chain complex activities in Parkinson's, Alzheimer's, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant imbalance, are thought to underlie defects in energy metabolism and induce cellular degeneration. The precise sequence of events in FRDA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain, associated with increased free radical generation and oxidative damage, may be considered possible mechanisms that compromise cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, that decreased expression of frataxin protein is associated with FRDA. Many approaches have been undertaken to understand FRDA, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to FRDA pathogenesis. Brains of FRDA patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress. In the central nervous system, heat shock protein (HSP) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment. The major neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease (HD) and FRDA are all associated with the presence of abnormal proteins. Among the various HSPs, HSP32, also known as heme oxygenase I (HO-1), has received considerable attention, as it has been recently demonstrated that HO-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. This may open up new perspectives in medicine, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms, such as the heat shock response, through nutritional antioxidants, pharmacological compounds or gene transduction, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.

Aging↗

PubMed2005

Oxidative modification and down-regulation of Pin1 in Alzheimer's disease hippocampus: A redox proteomics analysis.

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.

Aged, 80 and over↗

PubMed2005

Proteomic identification of proteins oxidized by Abeta(1-42) in synaptosomes: implications for Alzheimer's disease.

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.

Amyloid beta-Peptides↗

PubMed2005

In vivo protection of synaptosomes from oxidative stress mediated by Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride by the glutathione mimetic tricyclodecan-9-yl-xanthogenate.

D609 (tricyclodecan-9-yl-xanthogenate) is a phosphatidylcholine-specific phospholipase C inhibitor that also has been reported to protect rodents against oxidative damage caused by lethal doses of ionizing radiation. We previously showed that D609 mimics glutathione. D609 has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies from our laboratory have also shown that D609 reduces the Alzheimer amyloid beta-peptide (1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture. The present study was undertaken to test the hypothesis that D609 would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes isolated from gerbils, previously injected intraperitoneally (ip) with D609, were treated with the oxidants Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH), which produce free radicals. Synaptosomes isolated from the gerbils ip injected with D609 and treated with Fe2+/H2O2 or AAPH showed significant reduction in reactive oxygen species, levels of protein carbonyl, protein-bound hydroxynonenal (a lipid peroxidation product), and 3-nitrotyrosine (another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to oxidative stress in synaptosomes isolated from gerbils that were injected with saline, but treated with Fe2+/H2O2 or AAPH. These results are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders.

Amidines↗

PubMed2005

Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis.

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.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis.

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.

Aldehydes↗

PubMed2005

Gamma-glutamylcysteine ethyl ester protection of proteins from Abeta(1-42)-mediated oxidative stress in neuronal cell culture: a proteomics approach.

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.

Amyloid beta-Peptides↗

PubMed2005

Gamma-glutamylcysteine ethyl ester-induced up-regulation of glutathione protects neurons against Abeta(1-42)-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease.

Glutathione (GSH) is an important endogenous antioxidant found in millimolar concentrations in the brain. GSH levels have been shown to decrease with aging. Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging and oxidative stress. Abeta(1-42) has been shown to induce oxidative stress and has been proposed to play a central role in the oxidative damage detected in AD brain. It has been shown that administration of gamma-glutamylcysteine ethyl ester (GCEE) increases cellular levels of GSH, circumventing the regulation of GSH biosynthesis by providing the limiting substrate. In this study, we evaluated the protective role of up-regulation of GSH by GCEE against the oxidative and neurotoxic effects of Abeta(1-42) in primary neuronal culture. Addition of GCEE to neurons led to an elevated mean cellular GSH level compared with untreated control. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine (BSO) led to a 98% decrease in total cellular GSH compared with control, which was returned to control levels by addition of GCEE. Taken together, these results suggest that GCEE up-regulates cellular GSH levels which, in turn, protects neurons against protein oxidation, loss of mitochondrial function, and DNA fragmentation induced by Abeta(1-42). These results are consistent with the notion that up-regulation of GSH by GCEE may play a viable protective role in the oxidative and neurotoxicity induced by Abeta(1-42) in AD brain.

Alzheimer Disease↗

PubMed2005

Proteomics analysis of human astrocytes expressing the HIV protein Tat.

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.

Analysis of Variance↗

PubMed2005

Proteomic analysis of oxidatively modified proteins induced by the mitochondrial toxin 3-nitropropionic acid in human astrocytes expressing the HIV protein tat.

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.

Astrocytes↗

PubMed2005

Acetylcarnitine induces heme oxygenase in rat astrocytes and protects against oxidative stress: involvement of the transcription factor Nrf2.

Efficient functioning of maintenance and repair processes seem to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, under control of several genes termed vitagenes. These include members of the heat shock protein system, and there is now evidence that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human conditions, including inflammation, neurodegenerative disorders, and aging. Among the various heat shock proteins, heme oxygenase-1 has received considerable attention; it has been recently demonstrated that heme oxygenase-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, we report here that treatment of astrocytes with acetyl-L-carnitine induces heme oxygenase-1 in a dose- and time-dependent manner and that this effect was associated with up-regulation of heat shock protein 60 as well as high expression of the redox-sensitive transcription factor Nrf2 in the nuclear fraction of treated cells. In addition, we show that addition of acetyl-L-carnitine to astrocytes, prior to proinflammatory lipopolysaccharide- and interferon-gamma-induced nitrosative stress, prevents changes in mitochondrial respiratory chain complex activity, protein nitrosation and antioxidant status induced by inflammatory cytokine insult. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases.

Acetylcarnitine↗

PubMed2005

Mitochondrial associated metabolic proteins are selectively oxidized in A30P alpha-synuclein transgenic mice--a model of familial Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra compacta. alpha-Synuclein is strongly implicated in the pathophysiology of PD because aggregated alpha-synuclein accumulates in the brains of subjects with PD, mutations in alpha-synuclein cause familial PD, and overexpressing mutant human alpha-synuclein (A30P or A53T) causes degenerative disease in mice or drosophila. The pathophysiology of PD is poorly understood, but increasing evidence implicates mitochondrial dysfunction and oxidative stress. To understand how mutations in alpha-synuclein contribute to the pathophysiology of PD, we undertook a proteomic analysis of transgenic mice overexpressing A30P alpha-synuclein to investigate which proteins are oxidized. We observed more than twofold selective increases in specific carbonyl levels of three metabolic proteins in brains of symptomatic A30P alpha-synuclein mice: carbonic anhydrase 2 (Car2), alpha-enolase (Eno1), and lactate dehydrogenase 2 (Ldh2). Analysis of the activities of these proteins demonstrates decreased functions of these oxidatively modified proteins in brains from the A30P compared to control mice. Our findings suggest that proteins associated with impaired energy metabolism and mitochondria are particularly prone to oxidative stress associated with A30P-mutant alpha-synuclein.

Alanine↗

PubMed2005

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders.

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.

Aging↗

PubMed2005

Free radical mediated oxidative stress and toxic side effects in brain induced by the anti cancer drug adriamycin: insight into chemobrain.

Adriamycin (ADR) is a chemotherapeutic agent useful in treating various cancers. ADR is a quinone-containing anthracycline chemotherapeutic and is known to produce reactive oxygen species (ROS) in heart. Application of this drug can have serious side effects in various tissues, including brain, apart from the known cardiotoxic side effects, which limit the successful use of this drug in treatment of cancer. Neurons treated with ADR demonstrate significant protein oxidation and lipid peroxidation. Patients under treatment with this drug often complain of forgetfulness, lack of concentration, dizziness (collectively called somnolence or sometimes called chemobrain). In this study, we tested the hypothesis that ADR induces oxidative stress in brain. Accordingly, we examined the in vivo levels of brain protein oxidation and lipid peroxidation induced by i.p. injection of ADR. We also measured levels of the multidrug resistance-associated protein (MRP1) in brain isolated from ADR- or saline-injected mice. MRP1 mediates ATP-dependent export of cytotoxic organic anions, glutathione S-conjugates and sulphates. The current results demonstrated a significant increase in levels of protein oxidation and lipid peroxidation and increased expression of MRP1 in brain isolated from mice, 72 h post i.p injection of ADR. These results are discussed with reference to potential use of this redox cycling chemotheraputic agent in the treatement of cancer and its chemobrain side effect in brain.

Aldehydes↗