Identification of novel N-terminal fragments of amyloid precursor protein in cerebrospinal fluid
ABSTRACT Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system. Two pathological hallmarks in the brain of AD patients are neurofibrillary tangles and senile plaques. The plaques consist mainly of beta-amyloid (Abeta) peptides that are produced from the amyloid precursor protein (APP), by sequential cleavage by beta- and gamma-secretase. Most previous studies have been focused on the C-terminal fragments of APP, where the Abeta sequence is localized. The purpose of this study was to search for N-terminal fragments of APP in cerebrospinal fluid (CSF) using mass spectrometry (MS). By using immunoprecipitation (IP) combined with matrix-assisted laser desorption/ionization time-of-flight MS as well as nanoflow liquid chromatography coupled to high resolution tandem MS we were able to detect and identify six novel N-terminal APP fragments [APP((18-119)), APP((18-121)), APP((18-122)), APP((18-123)), APP((18-124)) and APP((18-126))], having molecular masses of approximately 12 kDa. The presence of these APP derivatives in CSF was also verified by Western blot analysis. Two pilot studies using either IP-MS or Western blot analysis indicated slightly elevated levels of N-terminal APP fragments in CSF from AD patients compared with controls, which are in need of replications in independent and larger patient materials.
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- "For example, APP is cleaved by a-or b-secretase to produce ectodomain fragments (sAPPa or sAPPb) that are secreted into the extracellular milieu (Nunan and Small, 2000). Several studies have shown that sAPPa and APP can be cleaved, both in vitro and in vivo, to yield smaller N-terminal fragments (Small et al., 1994; Caswell et al., 1999: Esh et al., 2005; Nikolaev et al., 2009; Portelius et al., 2010; Jefferson et al., 2011; Vella and Cappai, 2012). The level of APP N-terminal fragments has been reported to increase during development as cells differentiate into neurons (Vella and Cappai, 2012). "
ABSTRACT: The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited by a short-acyl-chain PIP analogue, and N-APP did not bind to glial cells which also possessed PI(4,5)P2 on the cell surface. The data are explained by a model in which N-APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N-APP. Our data provide further support for the idea that N-APP may be an important mediator of APP's biological activity. © 2014 Wiley Periodicals, Inc.Journal of Neuroscience Research 11/2014; 92(11). DOI:10.1002/jnr.23422 · 2.73 Impact Factor
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ABSTRACT: "clinical NEUroPROteomics of neurodegenerative diseases" (cNEUPRO) is a Specific Targeted Research Project (STREP) within the sixth framework program of the European Commission dedicated to the search for novel biomarker candidates for Alzheimer's disease and other neurodegenerative diseases. The ultimate goal of cNEUPRO is to identify one or more valid biomarker(s) in blood and CSF applicable to support the early and differential diagnosis of dementia disorders. The consortium covers all steps required for the discovery of novel biomarker candidates such as acquisition of high quality CSF and blood samples from relevant patient groups and controls, analysis of body fluids by various methods, and finally assay development and assay validation. Here we report the standardized procedures for diagnosis and preanalytical sample-handling within the project, as well as the status of the ongoing research activities and some first results.09/2010; 2010. DOI:10.4061/2010/548145
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ABSTRACT: Tryptophanyl-tRNA synthetase (TrpRS) catalyzes tryptophanyl-tRNAtrp formation. At concentrations exceeding tryptophan, tryptamine inhibits TrpRS. This leads in tryptophanyl-tRNA deficiency and synthesis of aberrant proteins. Tryptamine presents in food and crosses blood-brain barrier. The purpose of this study is to test the hypothesis that tryptamine-induced changes in cell and animal models correlate with Alzheimer's disease (AD) manifestations. Tryptamine prevented growth of human neuroblastoma. Epithelioids recovered growth in tryptamine-free medium, while neuroblasts died. Tryptamine induced epithelioid differentiation forming synaptic vesicles, neuritic contacts, and TrpRS+ axons in stable sublines. A fraction of epithelioids was adhered to satellite cells via trypsin-resistant interdigitating junctions. Tryptamine stimulated satellite division and differentiation into neurons, transitional cell variants and neuroblasts able to repopulate. Both tryptamine-inhibited and hypoxia-downregulated TrpRS translocates into cytoplasmic extensions. TrpRS is secreted into extracellular space as a free protein or within vesicles extended from cytoplasm and then pinched-off from plasma membrane of tryptamine-treated cells. Extracellular vesicles fuse in congophilic TrpRS+ plaques in tryptamine-treated culture and AD brain. TrpRS prominent immunoreactivity is associated with plasma and vesicle membranes of satellites and AD brain degenerated neurons. Tryptamine-modified mouse brain expresses amyloid and abnormal filaments in extracellular and neuronal plasma membrane vesicles. Radiolabeled tryptamine, tryptophan and serotonin uptake was 10-fold lower in tryptamine-resistant compared to tryptamine-sensitive cells. In both variants, tryptamine uptake exceeded tryptophan uptake within 2-h assuring TrpRS inhibition. Here, tryptophanyl-tRNAtrp deficiency implicates in both neurite growth and termination/collapse. Neurite growth termination prompts TrpRS+ vesicularization. TrpRS+ vesicles contribute in neuronal fragmentation and fibrillar-vesicular congophilic plaques in AD brain.Journal of Alzheimer's disease: JAD 05/2011; 26(2):263-98. DOI:10.3233/JAD-2011-110176 · 4.15 Impact Factor