Jon B. Klein

Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: An approach to understand pathological and biochemical alterations in AD

Alzheimers 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 Alzheimers disease.

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.

An increase in S-glutathionylated proteins in the Alzheimer's disease inferior parietal lobule, a proteomics approach

Alzheimers disease (AD) is a neurodegenerative disorder characterized by neurofibrillary tangles, senile plaques, and loss of synapses. Many studies support the notion that oxidative stress plays an important role in AD pathogenesis. Previous studies from our laboratory employed redox proteomics to identify oxidatively modified proteins in the AD inferior parietal lobule (IPL) and hippocampus. The proteins were consistent with biochemical or pathological alterations in AD and have been central to further investigations of the disease. The present study focused on the identification of specific targets of protein S‐glutathionylation in AD and control IPL by using a redox proteomics approach. For AD IPL, we identified deoxyhemoglobin, α‐crystallin B, glyceraldehyde phosphate dehydrogenase (GAPDH), and α‐enolase as significantly S‐glutathionylated relative to these brain proteins in control IPL. GAPDH and α‐enolase were also shown to have reduced activity in the AD IPL. This study demonstrates that specific proteins are sensitive to S‐glutathionylation, which most likely is due to their sensitivity to cysteine oxidation initiated by the increase in oxidative stress in the AD brain.

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.

Redox proteomics identification of oxidatively modified brain proteins in inherited Alzheimer's disease: An initial assessment

OBJECTIVE To identify oxidatively modified proteins in brains of persons with inherited Alzheimers 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), gamma-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.

Protective effect of D609 against amyloid-beta1-42-induced oxidative modification of neuronal proteins: redox proteomics study.

Oxidative stress has been implicated in the pathophysiology of a number of diseases, including neurodegenerative disorders such as Alzheimers disease (AD), a neurodegenerative disorder associated with cognitive decline and enhanced oxidative stress. Amyloid‐beta peptide1–42 (Aβ1–42), one of the main component of senile plaques, can induce in vitro and in vivo oxidative damage to neuronal cells through its ability to produce free radicals. The aim of this study was to investigate the protective effect of the xanthate D609 on Aβ1–42‐induced protein oxidation by using a redox proteomics approach. D609 was recently found to be a free radical scavenger and antioxidant. In the present study, rat primary neuronal cells were pretreated with 50 μM of D609, followed by incubation with 10 μM Aβ1–42 for 24 hr. In the cells treated with Aβ1–42 alone, four proteins that were significantly oxidized were identified: glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, malate dehydrogenase, and 14‐3‐3 zeta. Pretreatment of neuronal cultures with D609 prior to Aβ1–42 protected all the identified oxidized proteins in the present study against Aβ1–42‐mediated protein oxidation. Therefore, D609 may ameliorate the Aβ1–42‐induced oxidative modification. We discuss the implications of these Aβ1–42‐mediated oxidatively modified proteins for AD pathology and for potential therapeutic intervention in this dementing disorder.

Proteomic Analysis of Human Neutrophils

Proteomics is the study of the set of proteins, or proteome, expressed by a cell under specific conditions. Proteomics methodology consists of protein extraction, protein separation, and protein identification. Currently, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry are the most widespread methods for proteomic studies. The recent introduction of precast immobilized pH gradient gel strips, precast gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and well-designed electrophoresis equipment has made 2DE a highly reproducible and relatively simple method for protein separation. Inherent limitations of the procedure, however, require approaches in sample preparation that may be cell- or tissue-dependent. This chapter describes a methodology for proteomic analysis of human neutrophils and discusses its applications.