Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection
ABSTRACT The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins co-isolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis.
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ABSTRACT: Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a non-phosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 non-redundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain.This article is protected by copyright. All rights reservedProteomics 10/2014; 15(2-3). DOI:10.1002/pmic.201400171 · 3.97 Impact Factor
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ABSTRACT: The senile plaque is a hallmark lesion of Alzheimer disease (AD). We compared, without a priori, the lipidome of the senile plaques and of the adjacent plaque-free neuropil. The analysis by liquid chromatography coupled with electrospray ionization mass spectrometry revealed that laser microdissected senile plaques were enriched in saturated ceramides Cer(d18:1/18:0) and Cer(d18:1/20:0) by 33 and 78 % respectively with respect to the surrounding neuropil. This accumulation of ceramides was not explained by their affinity for Aβ deposits: no interaction between ceramide-liposomes and Aβ fibrils was observed in vitro by surface plasmon resonance and fluorescent ceramide-liposomes showed no affinity for the senile plaques in AD brain tissue. Accumulation of ceramides could be, at least partially, the result of a local production by acid and neutral sphingomyelinase that we found to be present in the corona of 78 % and 82 % of the senile plaque.Neurobiology of Disease 01/2014; 65. DOI:10.1016/j.nbd.2014.01.010 · 5.20 Impact Factor
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ABSTRACT: Late-onset Alzheimer's disease (LOAD) is the most common neurodegenerative disorder in older adults, affecting over 50% of those over age 85. Aging is the most important risk factor for the development of LOAD. Aging is associated with the decrease in the ability of cells to cope with cellular stress, especially protein aggregation. Here we describe how the process of aging affects pathways that control the processing and degradation of abnormal proteins including amyloid-β (Aβ). Genetic association studies in LOAD have successfully identified a large number of genetic variants involved in the development of the disease. However, there is a gap in understanding the interconnections between these pathomolecular events that prevent us from discovering therapeutic targets. We propose novel, pertinent links to elucidate how the biology of aging affects the sequence of events in the development of LOAD. Furthermore we analyze and synthesize the molecular-pathologic-clinical correlations of the aging process, involving the HSF1 and FOXO family pathways, Aβ metabolic pathway, and the different clinical stages of LOAD. Our new model postulates that the aging process would precede Aβ accumulation, and attenuation of HSF1 is an "upstream" event in the cascade that results in excess Aβ and synaptic dysfunction, which may lead to cognitive impairment and/or trigger "downstream" neurodegeneration and synaptic loss. Specific host factors, such as the activity of FOXO family pathways, would mediate the response to Aβ toxicity and the pace of progression toward the clinical manifestations of AD.Journal of Alzheimer's disease: JAD 12/2013; 40(1). DOI:10.3233/JAD-131544 · 3.61 Impact Factor