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.

Increased pulmonary arteriolar tone associated with lung oxidative stress and nitric oxide in a mouse model of Alzheimer's disease.

Vascular dysfunction and decreased cerebral blood flow are linked to Alzheimer's disease (AD). Loss of endothelial nitric oxide (NO) and oxidative stress in human cerebrovascular endothelium increase expression of amyloid precursor protein (APP) and enhance production of the Aß peptide, suggesting that loss of endothelial NO contributes to AD pathology. We hypothesize that decreased systemic NO bioavailability in AD may also impact lung microcirculation and induce pulmonary endothelial dysfunction. The acute effect of NO synthase (NOS) inhibition on pulmonary arteriolar tone was assessed in a transgenic mouse model (TgAD) of AD (C57BL/6-Tg(Thy1-APPSwDutIowa)BWevn/Mmjax) and age-matched wild-type controls (C57BL/6J). Arteriolar diameters were measured before and after the administration of the NOS inhibitor, L-NAME Lung superoxide formation (DHE) and formation of nitrotyrosine (3-NT) were assessed as indicators of oxidative stress, inducible NOS (iNOS) and tumor necrosis factor alpha (TNF-α) expression as indicators of inflammation. Administration of L-NAME caused either significant pulmonary arteriolar constriction or no change from baseline tone in wild-type (WT) mice, and significant arteriolar dilation in TgAD mice. DHE, 3-NT, TNF-α, and iNOS expression were higher in TgAD lung tissue, compared to WT mice. These data suggest L-NAME could induce increased pulmonary arteriolar tone in WT mice from loss of bioavailable NO In contrast, NOS inhibition with L-NAME had a vasodilator effect in TgAD mice, potentially caused by decreased reactive nitrogen species formation, while significant oxidative stress and inflammation were present. We conclude that AD may increase pulmonary microvascular tone as a result of loss of bioavailable NO and increased oxidative stress. Our findings suggest that AD may have systemic microvascular implications beyond central neural control mechanisms.

Target ablation-induced regulation of macrophage recruitment into the olfactory epithelium of Mip-1alpha-/- mice and restoration of function by exogenous MIP-1alpha.

The chemokine macrophage inflammatory protein (MIP)-1alpha recruits macrophages to sites of epithelial remodeling. We showed previously that mRNA and protein levels of MIP-1alpha in the olfactory epithelium (OE) increased significantly at 3 days after bilateral olfactory bulbectomy (OBX). The first aim of this study was to investigate the effect of the absence of MIP-1alpha on macrophage recruitment to the OE 3 days after OBX in Mip-1alpha(-/-) mice compared with C57BL/6 mice and to test whether chemokine function could be restored by MIP-1alpha protein injection into Mip-1alpha(-/-) mice. OBX was performed on C57BL/6 and Mip-1alpha(-/-) mice. The mice received six subcutaneous injections at 12-h intervals of either 10 mug/ml MIP-1alpha protein in carrier or carrier only. Macrophage recruitment was evaluated with antibodies to CD68 for all macrophages and F4/80 for activated macrophages. Compared with C57BL/6 mice, at 3 days post-OBX the numbers of CD68(+) and F4/80(+) macrophages were significantly lower in carrier-injected Mip-1alpha(-/-) mice and were comparable in MIP-1alpha protein-injected Mip-1alpha(-/-) mice. The second aim was to determine the identity of genes regulated at 3 days post-OBX in the OE of carrier-injected Mip-1alpha(-/-) mice compared with carrier-injected C57BL/6 mice. Total RNA from the OE was hybridized to Affymetrix microarrays. A number of chemokine-, cytokine-, and growth factor-related genes were significantly regulated in the Mip-1alpha(-/-) mice and were restored in MIP-1alpha protein-injected Mip-1alpha(-/-) mice. The results illustrated that MIP-1alpha played a key role in recruitment of macrophages to the OE and provided insight into the genomic regulation involved in OE remodeling.

Cross-kingdom sequence similarities between human micro-RNAs and plant viruses.

Micro-RNAs regulate the expression of cellular and tissue phenotypes at a post-transcriptional level through a complex process involving complementary interactions between micro-RNAs and messenger-RNAs. Similar nucleotide interactions have been shown to occur as cross-kingdom events; for example, between plant viruses and plant micro-RNAs and also between animal viruses and animal micro-RNAs. In this study, this view is expanded to look for cross-kingdom similarities between plant virus and human micro-RNA sequences. A method to identify significant nucleotoide sequence similarities between plant viruses and hsa micro-RNAs was created. Initial analyses demonstrate that plant viruses contain nucleotide sequences which exactly match the seed sequences of human micro-RNAs in both parallel and anti-parallel directions. For example, the bean common mosaic virus strain NL4 from Colombia contains sequences that match exactly the seed sequence for micro-RNA of the hsa-mir-1226 in the parallel direction, which suggests a cross-kingdom conservation. Similarly, the rice yellow stunt viral cRNA contains a sequence that is an exact match in the anti-parallel direction to the seed sequence of hsa-micro-RNA let-7b. The functional implications of these results need to be explored. The finding of these cross-kingdom sequence similarities is a useful starting point in support of bench level investigations.

Humans have antibodies against a plant virus: evidence from tobacco mosaic virus.

Tobacco mosaic virus (TMV), a widespread plant pathogen, is found in tobacco (including cigarettes and smokeless tobacco) as well as in many other plants. Plant viruses do not replicate or cause infection in humans or other mammals. This study was done to determine whether exposure to tobacco products induces an immune response to TMV in humans. Using a sandwich ELISA assay, we detected serum anti-TMV antibodies (IgG, IgG1, IgG3, IgG4, IgA, and IgM) in all subjects enrolled in the study (20 healthy smokers, 20 smokeless-tobacco users, and 20 non-smokers). Smokers had a higher level of serum anti-TMV IgG antibodies than non-smokers, while the serum level of anti-TMV IgA from smokeless tobacco users was lower than smokers and non-smokers. Using bioinformatics, we also found that the human protein TOMM40L (an outer mitochondrial membrane 40 homolog--like translocase) contains a strong homology of six contiguous amino acids to the TMV coat protein, and TOMM40L peptide exhibited cross-reactivity with anti-TMV antibodies. People who smoke cigarettes or other tobacco products experience a lower risk of developing Parkinson's disease, but the mechanism by which this occurs is unclear. Our results showing molecular mimicry between TMV and human TOMM40L raise the question as to whether TMV has a potential role in smokers against Parkinson's disease development. The potential mechanisms of molecular mimicry between plant viruses and human disease should be further explored.

Aquaporin 4 molecular mimicry and implications for neuromyelitis optica.

Neuromyelitis optica (NMO) is associated with antibodies to aquaporin 4 (AQP4). We hypothesized that antibodies to AQP4 can be triggered by exposure to environmental proteins. We compared human AQP4 to plant and bacterial proteins to investigate the occurrence of significantly similar structures and sequences. High similarity to a known epitope for NMO-IgG, AQP4(207-232), was observed for corn ZmTIP4-1. NMO and non-NMO sera were assessed for reactivity to AQP4(207-232) and the corn peptide. NMO patient serum showed reactivity to both peptides as well as to plant tissue. These findings warrant further investigation into the role of the environment in NMO etiology.

Antioxidant therapies for traumatic brain injury.

Free radical-induced oxidative damage reactions, and membrane lipid peroxidation (LP), in particular, are among the best validated secondary injury mechanisms in preclinical traumatic brain injury (TBI) models. In addition to the disruption of the membrane phospholipid architecture, LP results in the formation of cytotoxic aldehyde-containing products that bind to cellular proteins and impair their normal functions. This article reviews the progress of the past three decades in regard to the preclinical discovery and attempted clinical development of antioxidant drugs designed to inhibit free radical-induced LP and its neurotoxic consequences via different mechanisms including the O(2)(*-) scavenger superoxide dismutase and the lipid peroxidation inhibitor tirilazad. In addition, various other antioxidant agents that have been shown to have efficacy in preclinical TBI models are briefly presented, such as the LP inhibitors U83836E, resveratrol, curcumin, OPC-14177, and lipoic acid; the iron chelator deferoxamine and the nitroxide-containing antioxidants, such as alpha-phenyl-tert-butyl nitrone and tempol. A relatively new antioxidant mechanistic strategy for acute TBI is aimed at the scavenging of aldehydic LP byproducts that are highly neurotoxic with "carbonyl scavenging" compounds. Finally, it is proposed that the most effective approach to interrupt posttraumatic oxidative brain damage after TBI might involve the combined treatment with mechanistically complementary antioxidants that simultaneously scavenge LP-initiating free radicals, inhibit LP propagation, and lastly remove neurotoxic LP byproducts.

Lipid peroxidation-derived reactive aldehydes directly and differentially impair spinal cord and brain mitochondrial function.

Mitochondrial bioenergetic dysfunction in traumatic spinal cord and brain injury is associated with post-traumatic free radical-mediated oxidative damage to proteins and lipids. Lipid peroxidation by-products, such as 4-hydroxy-2-nonenal and acrolein, can form adducts with proteins and exacerbate the effects of direct free radical-induced protein oxidation. The aim of the present investigation was to determine and compare the direct contribution of 4-hydroxy-2-nonenal and acrolein to spinal cord and brain mitochondrial dysfunction. Ficoll gradient-isolated mitochondria from normal rat spinal cords and brains were treated with carefully selected doses of 4-hydroxy-2-nonenal or acrolein, followed by measurement of complex I- and complex II-driven respiratory rates. Both compounds were potent inhibitors of mitochondrial respiration in a dose-dependent manner. 4-Hydroxy-2-nonenal significantly compromised spinal cord mitochondrial respiration at a 0.1-muM concentration, whereas 10-fold greater concentrations produced a similar effect in brain. Acrolein was more potent than 4-hydroxy-2-nonenal, significantly decreasing spinal cord and brain mitochondrial respiration at 0.01 muM and 0.1 muM concentrations, respectively. The results of this study show that 4-hydroxy-2-nonenal and acrolein can directly and differentially impair spinal cord and brain mitochondrial function, and that the targets for the toxic effects of aldehydes appear to include pyruvate dehydrogenase and complex I-associated proteins. Furthermore, they suggest that protein modification by these lipid peroxidation products may directly contribute to post-traumatic mitochondrial damage, with spinal cord mitochondria showing a greater sensitivity than those in brain.

Cellular and molecular characterization of oxidative stress in olfactory epithelium of Harlequin mutant mouse.

Oxidative stress in the olfactory system is a major factor associated with age-related olfactory impairment, although the mechanisms by which this occurs are not completely understood. The Harlequin mutant mouse (Hq/Y), which carries an X-linked recessive mutation in the Aifm1 gene, is a model of progressive oxidative stress-induced neurodegeneration in the cerebellum and retina. To determine whether the Hq/Y mutant mouse is a suitable model of oxidative stress-associated olfactory aging, we investigated cellular and molecular changes in the olfactory epithelium (OE) and olfactory bulb (OB) of 6-month-old male Hq/Y mice compared to those in sex-matched littermate controls (+/Y) and in age- and sex-matched C57BL/6 mice. Immunoreactivity for apoptosis-inducing factor, the protein product of Aifm1, was localized in mature olfactory sensory neurons (mOSNs) in +/Y mice but was rarely detected in Hq/Y mice. Hq/Y mice also exhibited increased lipofuscin autofluorescence and increased immunoreactivity for an oxidative DNA/RNA damage marker in mOSNs and in mitral/tufted cells in the OB and an increased number of cleaved caspase-3 immunoreactive apoptotic cells in the OE. Microarray analysis demonstrated that Aifm1 expression was down-regulated by 80% in the OE of Hq/Y mice compared to that in +/Y mice. Most significantly, regulated genes were classified into functional categories of cell signaling/apoptosis/cell cycle, oxidative stress/aging, and cytoskeleton/extracellular matrix/transport-associated. Analysis with EASE software indicated that the functional categories significantly overrepresented in Hq/Y mice included up-regulated mitochondrial genes and down-regulated cytoskeletal organization- and neurogenesis-related genes. Our results strongly support the Hq/Y mutant mouse being a novel model for mechanistic studies of oxidative stress-associated olfactory aging.

Oxidative stress in the aging murine olfactory bulb: redox proteomics and cellular localization.

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.

Proteomic identification of differentially expressed proteins in the aging murine olfactory system and transcriptional analysis of the associated genes.

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.

Temporal profiling of gene expression during neurogenesis and remodeling in the olfactory epithelium at short intervals after target ablation.

Neurogenesis in the olfactory epithelium (OE) is induced by olfactory bulbectomy (OBX), which effectively axotomizes olfactory sensory neurons (OSNs) and removes their synaptic targets, resulting in apoptosis. We used Affymetrix high-density oligonucleotide arrays to investigate changes in gene expression during initiation of signaling in pathways that regulate apoptosis and neurogenesis in the murine OE at 2, 8, 16, and 48 hr after bilateral OBX compared to that in sham-operated controls. We focused on regulation of a defined set of genes associated with apoptosis, stem/progenitor cell regulation, and cell cycle progression because of the activation of these processes in OE degeneration and remodeling after OBX. After data scrubbing and categorical analysis, one-way analysis of variance identified 72 genes (4.9% of the present known genes) as being regulated significantly (P < 0.05) at one or more points; 50 were defined as regulated differentially with the false discovery rate at 10%. Significant changes in gene expression occurred in all categories as early as 2 hr post-OBX, with the greatest number of differentially regulated genes at 16 and 48 hr. Hierarchical cluster analysis and correlation coefficients were used to identify similarities in patterns of gene expression changes within and across categories. Validation was carried out with SuperArray macroarrays and real-time RT-PCR. Our results confirmed the participation of many genes in known signaling pathways and identified changes in the expression of 42 genes not identified previously as participating in apoptosis and neurogenesis in the OE. Additionally, our analyses indicated the early involvement of genes regulating cytoskeletal reorganization and angiogenesis in the response to OBX. These studies are an important first step in defining early time-dependent changes in gene expression after target ablation that lead to neurogenesis in the olfactory sensory epithelium.