Renã A. Sowell

In vivo oxidative stress in brain of Alzheimer disease transgenic mice: Requirement for methionine 35 in amyloid β-peptide of APP

Numerous studies have demonstrated oxidative damage in the central nervous system in subjects with Alzheimer disease and in animal models of this dementing disorder. In this study, we show that transgenic mice modeling Alzheimer disease-PDAPP mice with Swedish and Indiana mutations in the human amyloid precursor protein (APP)-develop oxidative damage in brain, including elevated levels of protein oxidation (indexed by protein carbonyls and 3-nitrotyrosine) and lipid peroxidation (indexed by protein-bound 4-hydroxy-2-nonenal). This oxidative damage requires the presence of a single methionine residue at position 35 of the amyloid beta-peptide (Abeta), because all indices of oxidative damage in brain were completely prevented in genetically and age-matched PDAPP mice with an M631L mutation in APP. No significant differences in the levels of APP, Abeta(1-42), and Abeta(1-40) or in the ratio Abeta(1-42)/Abeta(1-40) were found, suggesting that the loss of oxidative stress in vivo in the brain of PDAPP(M631L) mice results solely from the mutation of the Met35 residue to Leu in the Abeta peptide. However, a marked reduction in Abeta-immunoreactive plaques was observed in the M631L mice, which instead displayed small punctate areas of nonplaque immunoreactivity and a microglial response. In contrast to the requirement for Met at residue 35 of the Abeta sequence (M631 of APP) for oxidative damage, indices of spatial learning and memory were not significantly improved by the M631L substitution. Furthermore, a genetically matched line with a different mutation-PDAPP(D664A)-showed the reverse: no reduction in oxidative damage but marked improvement in memory. This is the first in vivo study to demonstrate the requirement for Abeta residue Met35 for oxidative stress in the brain of a mammalian model of Alzheimer disease. However, in this specific transgenic mouse model of AD, oxidative stress is neither required nor sufficient for memory abnormalities.

Proteomics in animal models of Alzheimer's and Parkinson's diseases

The risk of developing neurodegenerative disorders such as Alzheimers disease (AD) and Parkinsons disease (PD) increases with age. AD and PD are the two most common neurodegenerative diseases that currently affect millions of persons within the United States population. While many clues about the mechanisms of these disorders have been uncovered, to date, the molecular mechanisms associated with the cause of these diseases are not completely understood. Furthermore, there are no available cures or preventive treatments for either disorder. Animal models of AD and PD, though not perfect, offer a means to gain knowledge of the basic biochemistry associated with these disorders and with drug efficacy. The field of proteomics which focuses on identifying the dynamic nature of the protein content expressed within a particular cell, tissue, or organism, has provided many insights into these disturbing disorders. Proteomic studies have revealed many pathways that are associated with disease pathogenesis and that may lead to the development of potential therapeutic targets. This review provides a discussion of key findings from AD and PD proteomics-based studies in various animal models of disease.

Peptides and proteins in plasma and cerebrospinal fluid as biomarkers for the prediction, diagnosis, and monitoring of therapeutic efficacy of Alzheimer's disease

Alzheimers disease (AD) affects millions of persons worldwide. Earlier detection and/or diagnosis of AD would permit earlier intervention, which conceivably could delay progression of this dementing disorder. In order to accomplish this goal, reliable and specific biomarkers are needed. Biomarkers are multidimensional and have the potential to aid in various facets of AD such as diagnostic prediction, assessment of disease stage, discrimination from normally cognitive controls as well as other forms of dementia, and therapeutic efficacy of AD drugs. To date, biomarker research has focused on plasma and cerebrospinal fluid (CSF), two bodily fluids believed to contain the richest source of biomarkers for AD. CSF is the fluid surrounding the central nervous system (CNS), and is the most indicative obtainable fluid of brain pathology. Blood plasma contains proteins that affect brain processes from the periphery, as well as proteins/peptides exported from the brain; this fluid would be ideal for biomarker discovery due to the ease and non-invasive process of sample collection. However, it seems reasonable that biomarker discovery will result in combinations of CSF, plasma, and other fluids such as urine, to serve the aforementioned purposes. This review focuses on proteins and peptides identified from CSF, plasma, and urine that may serve as biomarkers in AD.

Quantitative Proteomics of a Presymptomatic A53T α-Synuclein Drosophila Model of Parkinson Disease

A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T α-synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 (∼71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T α-synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP synthase are known to be directly involved in A53T α-synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, manganese-superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature-supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicated that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicated that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P α-synuclein Drosophila model (Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Protein expression in a Drosophila model of Parkinsons disease. J. Proteome Res. 6, 348–357; Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Lifetime proteomic profiling of an A30P α-synuclein Drosophila model of Parkinsons disease. J. Proteome Res. 6, 3729–3738) that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms.

The wheat germ agglutinin-fractionated proteome of subjects with Alzheimer's disease and mild cognitive impairment hippocampus and inferior parietal lobule: Implications for disease pathogenesis and progression

Lectin affinity chromatography is a powerful separation technique that fractionates proteins by selectively binding to specific carbohydrate moieties characteristic of protein glycosylation type. Wheat germ agglutinin (WGA) selectively binds terminal N‐acetylglucosamine (O‐GlcNAc) and sialic acid moieties characteristic of O‐linked glycosylation. The current study utilizes WGA affinity chromatography to fractionate proteins from hippocampus and inferior parietal lobule (IPL) from subjects with Alzheimers disease (AD) and arguably its earliest form, mild cognitive impairment (MCI). Proteins identified by proteomics that were fractionated from MCI and AD hippocampus by WGA affinity chromatography with altered levels compared with age‐matched controls included GP96, γ‐enolase, glutamate dehydrogenase, glucosidase IIα, 14‐3‐3ϵ, 14‐3‐3γ, 14‐3‐3ζ, tropomyosin‐2, calmodulin 2, gelsolin, β‐synuclein, α1‐antichymotrypsin, and dimethylguanosine tRNA methyltransferase. Proteins identified by proteomics that were fractionated from MCI and AD IPL by WGA affinity chromatography showing altered levels compared with age‐matched controls included protein disulfide isomerase, calreticulin, and GP96. The proteins described in this study are involved in diverse processes, including glucose metabolism, endoplasmic reticulum (ER) functions, chaperoning, cytoskeletal assembly, and proteolysis, all of which are affected in AD. This study, the first to use proteomics to identify WGA‐fractionated proteins isolated from brains from subjects with MCI and AD, provides additional information about the active proteome of the brain throughout AD progression.

Development of a high-throughput IMS-IMS-MS approach for analyzing mixtures of biomolecules.

A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be +/-7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 h. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed.

Nitrated Proteins in the Progression of Alzheimer’s Disease: A Proteomics Comparison of Mild Cognitive Impairment and Alzheimer’s Disease Brain

Oxidative stress and nitrosative stress have been reported to play important roles in the pathogenesis of a number of diseases including neurodegenerative diseases, cancer, ischemia, etc. Reactive nitrogen species are highly reactive and unstable. One of the best ways to quantify the amount of nitrosative stress is to measure the levels of 3-nitrotyrosine level. In addition, by using proteomics selective targets of protein nitration can be identified. In this chapter we discuss the roles of proteomics-identified nitrated brain proteins to the pathology of both mild cognitive impairment and Alzheimer’s disease. The identity of these nitrated proteins improves understanding of the role of nitrosative stress in the pathogenesis and progression of disease from MCI to AD. Such studies could also help in early detection and may provide therapeutic targets for early treatment that may slow disease progression.