An investigation of the molecular mechanisms engaged before and after the development of Alzheimer disease neuropathology in Down syndrome: a proteomics approach.

Down syndrome (DS) is one of the most common causes of intellectual disability, owing to trisomy of all or part of chromosome 21. DS is also associated with the development of Alzheimer disease (AD) neuropathology after the age of 40 years. To better clarify the cellular and metabolic pathways that could contribute to the differences in DS brain, in particular those involved in the onset of neurodegeneration, we analyzed the frontal cortex of DS subjects with or without significant AD pathology in comparison with age-matched controls, using a proteomics approach. Proteomics represents an advantageous tool to investigate the molecular mechanisms underlying the disease. From these analyses, we investigated the effects that age, DS, and AD neuropathology could have on protein expression levels. Our results show overlapping and independent molecular pathways (including energy metabolism, oxidative damage, protein synthesis, and autophagy) contributing to DS, to aging, and to the presence of AD pathology in DS. Investigation of pathomechanisms involved in DS with AD may provide putative targets for therapeutic approaches to slow the development of AD.

Involvement of oxidative stress in occurrence of relapses in multiple sclerosis: the spectrum of oxidatively modified serum proteins detected by proteomics and redox proteomics analysis.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Several evidences suggest that MS can be considered a multi-factorial disease in which both genetics and environmental factors are involved. Among proposed candidates, growing results support the involvement of oxidative stress (OS) in MS pathology. The aim of this study was to investigate the role of OS in event of exacerbations in MS on serum of relapsing-remitting (RR-MS) patients, either in relapsing or remitting phase, with respect to serum from healthy subjects. We applied proteomics and redox proteomics approaches to identify differently expressed and oxidatively modified proteins in the low-abundant serum protein fraction. Among differently expressed proteins ceruloplasmin, antithrombin III, clusterin, apolipoprotein E, and complement C3, were up-regulated in MS patients compared with healthy controls. Further by redox proteomics, vitamin D-binding protein showed a progressive trend of oxidation from remission to relapse, respect with controls. Similarly, the increase of oxidation of apolipoprotein A-IV confirmed that levels of OS are elevated with the progression of the disease. Our findings support the involvement of OS in MS and suggest that dysfunction of target proteins occurs upon oxidative damage and correlates with the pathology.

Antisense directed against PS-1 gene decreases brain oxidative markers in aged senescence accelerated mice (SAMP8) and reverses learning and memory impairment: a proteomics study.

Amyloid ß-peptide (Aß) plays a central role in the pathophysiology of Alzheimer's disease (AD) through the induction of oxidative stress. This peptide is produced by proteolytic cleavage of amyloid precursor protein (APP) by the action of ß- and γ-secretases. Previous studies demonstrated that reduction of Aß, using an antisense oligonucleotide (AO) directed against the Aß region of APP, reduced oxidative stress-mediated damage and prevented or reverted cognitive deficits in senescence-accelerated prone mice (SAMP8), a useful animal model for investigating the events related to Aß pathology and possibly to the early phase of AD. In the current study, aged SAMP8 were treated by AO directed against PS-1, a component of the γ-secretase complex, and tested for learning and memory in T-maze foot shock avoidance and novel object recognition. Brain tissue was collected to identify the decrease of oxidative stress and to evaluate the proteins that are differently expressed and oxidized after the reduction in free radical levels induced by Aß. We used both expression proteomics and redox proteomics approaches. In brain of AO-treated mice a decrease of oxidative stress markers was found, and the proteins identified by proteomics as expressed differently or nitrated are involved in processes known to be impaired in AD. Our results suggest that the treatment with AO directed against PS-1 in old SAMP8 mice reverses learning and memory deficits and reduces Aß-mediated oxidative stress with restoration to the normal condition and identifies possible pharmacological targets to combat this devastating dementing disease.

Cell cycle proteins in brain in mild cognitive impairment: insights into progression to Alzheimer disease.

Recent studies have demonstrated the re-emergence of cell cycle proteins in brain as patients progress from the early stages of mild cognitive impairment (MCI) into Alzheimer's disease (AD). Oxidative stress markers present in AD have also been shown to be present in MCI brain suggesting that these events occur in early stages of the disease. The levels of key cell cycle proteins, such as CDK2, CDK5, cyclin G1, and BRAC1 have all been found to be elevated in MCI brain compared to age-matched control. Further, peptidyl prolyl cis-trans isomerase (Pin1), a protein that plays an important role in regulating the activity of key proteins, such as CDK5, GSK3-ß, and PP2A that are involved in both the phosphorylation state of Tau and in the cell cycle, has been found to be oxidatively modified and downregulated in both AD and MCI brain. Hyperphosphorylation of Tau then results in synapse loss and the characteristic Tau aggregation as neurofibrillary tangles, an AD hallmark. In this review, we summarized the role of cell cycle dysregulation in the progression of disease from MCI to AD. Based on the current literature, it is tempting to speculate that a combination of oxidative stress and cell cycle dysfunction conceivably leads to neurodegeneration.

Lack of p53 affects the expression of several brain mitochondrial proteins: insights from proteomics into important pathways regulated by p53.

The tumor suppressor protein p53 has been described "as the guardian of the genome" for its crucial role in regulating the transcription of numerous genes responsible for cells cycle arrest, senescence, or apoptosis in response to various stress signals. Although p53 promotes longevity by decreasing the risk of cancer through activation of apoptosis or cellular senescence, several findings suggest that an increase of its activity may have deleterious effects leading to selected aspects of the aging phenotype and neurodegenerative diseases. There is the link between p53 and oxidative stress, the latter a crucial factor that contributes to neurodegenerative processes like Alzheimer disease (AD). In the present study, using a proteomics approach, we analyzed the impact of lack of p53 on the expression of several brain mitochondrial proteins involved in different pathways, and how lack of p53 may present a target to restore neuronal impairments. Our investigation on isolated brain mitochondria from p53((-/-)) mice also provides a better understanding of the p53-mitochondria relationship and its involvement in the development of many diseases.

Lack of p53 decreases basal oxidative stress levels in the brain through upregulation of thioredoxin-1, biliverdin reductase-A, manganese superoxide dismutase, and nuclear factor kappa-B.

AIMS: The basal oxidative and nitrosative stress levels measured in cytosol, mitochondria, and nuclei as well as in the whole homogenate obtained from the brain of wild type (wt) and p53 knockout [p53((-/-))] mice were evaluated. We hypothesized that the loss of p53 could trigger the activation of several protective mechanisms such as those involving thioredoxin-1 (Thio-1), the heme-oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system, manganese superoxide dismutase (MnSOD), the IkB kinase type ß (IKKß)/nuclear factor kappa-B (NF-kB), and the nuclear factor-erythroid 2 (NF-E2) related factor 2 (Nrf-2). RESULTS: A decrease of protein carbonyls, protein-bound 4-hydroxy-2-nonenal (HNE), and 3-nitrotyrosine (3-NT) was observed in the brain from p53((-/-)) mice compared with wt. Furthermore, we observed a significant increase of the expression levels of Thio-1, BVR-A, MnSOD, IKKß, and NF-kB. Conversely a significant decrease of Nrf-2 protein levels was observed in the nuclear fraction isolated from p53((-/-)) mice. No changes were found for HO-1. INNOVATION: This is the first study of basal oxidative/nitrosative stress in in vivo conditions of brain obtained from p53((-/-)) mice. New insights into the role of p53 in oxidative stress have been gained. CONCLUSION: We demonstrated, for the first time, that the lack of p53 reduces basal oxidative stress levels in mice brain. Due to the pivotal role that p53 plays during cellular stress response our results provide new insights into novel therapeutic strategies to modulate protein oxidation and lipid peroxidation having p53 as a target. The implications of this work are profound, particularly for neurodegenerative disorders.

Oxidative stress in HPV-driven viral carcinogenesis: redox proteomics analysis of HPV-16 dysplastic and neoplastic tissues.

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins.

Do proteomics analyses provide insights into reduced oxidative stress in the brain of an Alzheimer disease transgenic mouse model with an M631L amyloid precursor protein substitution and thereby the importance of amyloid-beta-resident methionine 35 in Alzheimer disease pathogenesis?

The single methionine (Met/M) residue of amyloid-beta (Aß) peptide, at position 35 of the 42-mer, has important relevance for Aß-induced oxidative stress and neurotoxicity. Recent in vivo brain studies in a transgenic (Tg) Alzheimer disease (AD) mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) (referred to as the J20 Tg mouse) demonstrated increased levels of oxidative stress. However, the substitution of the Met631 residue of APP to leucine (Leu/L) (M631L in human APP numbering, referred to as M631L Tg and corresponding to residue 35 of Aß1-42) resulted in no significant in vivo oxidative stress levels, thereby supporting the hypothesis that Met-35 of Aß contributes to oxidative insult in the AD brain. It is conceivable that oxidative stress mediated by Met-35 of Aß is important in regulating numerous downstream effects, leading to differential levels of relevant biochemical pathways in AD. Therefore, in the current study using proteomics, we tested the hypothesis that several brain proteins involved in pathways such as energy and metabolism, antioxidant activity, proteasome degradation, and pH regulation are altered in J20Tg versus M631L Tg AD mice.

Oxidative stress occurs early in Down syndrome pregnancy: A redox proteomics analysis of amniotic fluid.

PURPOSE: The present study aims to evaluate a set of oxidative stress biomarkers in the amniotic fluid (AF) of women carrying Down syndrome (DS) fetuses that could prove in vivo the early occurrence of oxidative damage in DS. EXPERIMENTAL DESIGN: To assess the extent of protein oxidation in DS AF, we measured protein carbonylation and protein-bound HNE by slot-blot analysis, total and oxidized GSH levels by enzymatic assay and heat shock proteins (HSPs) thioredoxin (Trx) induction by Western blot. Further, by a redox proteomics approach specific targets of protein carbonylation were identified. RESULTS: We found increased levels of oxidative stress, as indexed by increased protein oxidation, lipid peroxidation, reduction of GSH and Trx levels and induction of the HSP response. By a redox proteomics approach, we identified selective proteins which showed increased oxidation in DS fetuses compared with healthy controls. The identified proteins are involved in iron homeostasis (ceruloplasmin and transferin), lipid metabolism (zinc-α2-glycoprotein, retinol-binding protein 4 and apolipoprotein A1) and inflammation (complement C9, α-1B-glycoprotein, collagen α-1V chain) with critical relevance in the clinical outcome of DS. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that oxidative damage is an early event in the DS pathogenesis and might contribute to the development of deleterious DS phenotypes, including abnormal development and AD-like neuropathology.