Brain amyloid-beta fragment signatures in pathological ageing and Alzheimer's disease by hybrid immunoprecipitation mass spectrometry.

BACKGROUND: Senile plaques in Alzheimer's disease (AD) are composed of amyloid-ß (Aß), especially N-truncated forms including Aß4-42. These are thought to be neurotoxic. However, individuals may live for decades with biomarker evidence of cerebral ß-amyloidosis (positive amyloid PET imaging and/or low cerebrospinal fluid levels of the 42 amino acid form of Aß) without cognitive impairment. This condition may be termed pathological ageing (PA). OBJECTIVE: To investigate whether there is a difference in the cerebral Aß fragment pattern in brain specimens from non-demented (PA) and demented (AD) individuals expressing the full neuropathological triad of AD (senile plaques, neurofibrillary tangles and neurodegeneration). METHODS: We extracted Aß using formic acid and hybrid (6E10 and 4G8) immunoprecipitation from fresh-frozen temporal cortex tissue of 6 elderly individuals (mean age ± SD: 89 ± 3.5 years) with PA and 10 patients with AD (mean age ± SD: 72 ± 8.5 years). The full spectrum of Aß peptides was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. RESULTS: AD patients had generally more N-terminally truncated and pyroglutamate-modified Aß than PA patients, whereas PA patients had on average more Aß1-40 than AD patients. CONCLUSION: Senile plaques in AD may have an Aß fragment composition distinct from PA with more N-terminally and pyroglutamate-modified Aß peptides that may be linked to neurotoxicity.

Exploring Alzheimer molecular pathology in Down's syndrome cerebrospinal fluid.

BACKGROUND: Individuals with Down's syndrome (DS) develop early Alzheimer's disease (AD) with ß-amyloid (Aß) plaque pathology. The extra amyloid precursor protein (APP) gene copy in DS is believed to result in a 50% increase in Aß production, but it is unclear how this relates to the development of other AD hallmarks, including axonal degeneration and microglia cell activation, and to other neurological problems in DS, including disturbed sleep regulation. OBJECTIVE: To evaluate if cerebrospinal fluid (CSF) biomarkers for cerebral amyloidosis, axonal degeneration, microglial activation and sleep regulation were altered in young and old patients with DS, and if these biomarkers were related to altered Aß and APP metabolism, reflected by CSF levels of different Aß and APP peptides. METHODS: CSF from DS patients (n=12) and healthy controls (n=20) were analyzed for Aß peptides (Aß1-42, AßX-38/40/42), secreted APP species (sAPPα/ß), biomarkers for AD-like axonal degeneration [total tau (T-tau), phosphorylated tau], microglial activation (YKL-40, CC chemokine ligand 2) and orexin-A, which is a peptide involved in sleep regulation. We compared biomarker levels between groups and tested for relations between biomarkers, disease stage and age. RESULTS: Several of the markers were specifically increased in DS, including AßX-40, sAPPα and sAPPß. Οrexin-A was significantly decreased in DS and correlated with Aß and sAPP. Orexin-A decreased with age in DS, while T-tau and YKL-40 increased with age. CONCLUSION: Down's patients have increased APP and Aß production and increased microglial activation with age. The orexin-A metabolism is disturbed in DS and may be linked to APP and Aß production. Biomarker studies of DS may contribute to our understanding of the amyloidogenic and neurodegenerative process in AD.

A novel pathway for amyloid precursor protein processing.

Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid ß (Aß) peptide and the non-amyloidogenic in which APP is cut in the middle of the Aß domain thus precluding Aß formation. Using immunoprecipitation and mass spectrometry we have shown that Aß is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Aß1-40 and Aß1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Aß isoform pattern in CSF. Using this model, we determined changes in the Aß isoform pattern induced by α-, ß-, and γ-secretase inhibitor treatment. All isoforms longer than and including Aß1-17 were γ-secretase dependent whereas shorter isoforms were γ-secretase independent. These shorter isoforms, including Aß1-14 and Aß1-15, were reduced by treatment with α- and ß-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by α- and ß-secretase.

Characterization of amyloid beta peptides in cerebrospinal fluid by an automated immunoprecipitation procedure followed by mass spectrometry.

Pathogenic events in Alzheimer's disease are believed to involve an imbalance between the production and clearance of the neurotoxic 42 amino acid form of the beta-amyloid peptide (Abeta1-42). Although much is known about the production of Abeta1-42, many questions remain about its degradation. Here, we describe an optimized automated immunoprecipitation mass spectrometry method that enables accurate and rapid monitoring of the major Abeta isoforms in cerebrospinal fluid. Furthermore, we describe a technique of antibody immobilization, minimizing background signals. The identities of these Abeta products were confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoflow liquid chromatography and tandem mass spectrometry with a hybrid linear trap Fourier transform ion cyclotron resonance mass spectrometer. Finally, we report the finding of two novel Abeta peptides (Abeta2-17 and Abeta3-17).

Analysis of proteins from a glioma cell line by using micro-scale solution isoelectric focusing in combination with liquid chromatography/tandem mass spectrometry.

In this study we have investigated whether micro-solution isoelectric focusing (microsol-IEF) can be used as a pre-fractionation step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) and if extensive sample purification of the different fractions is required. We found that, in spite of the high concentrations of buffer and detergents, no clean up of the digested microsol-IEF fractions was necessary before analysis by LC/MS/MS. We also concluded that it is possible to identify at least twice as many proteins in a glioma cell lysate with the combination of microsol-IEF and LC/MS/MS than with LC/MS/MS alone. Furthermore, most of the proteins that were identified from one microsol-IEF fraction by using analytical narrow-range two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were also identified by LC/MS/MS. Finally, we used the combination of microsol-IEF and LC/MS/MS to compare two sample preparation methods for glioma cells and found that several nuclear, mitochondria, and endoplasmic reticulum proteins were only present in the sample that had been subjected to lipid extraction by incubating the homogenized cells in chloroform/methanol/water.