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.

The amyloid-ß degradation pattern in plasma--a possible tool for clinical trials in Alzheimer's disease.

Amyloid beta (Aß) is the main component of plaques, the central neuropathological hallmark in Alzheimer's disease (AD). Aß is derived from the amyloid precursor protein (APP) by ß- and γ-secretase-mediated cleavages. A large number of Aß peptides are found in cerebrospinal fluid and these peptides are produced in specific metabolic pathways, which are important for diagnosis, in drug development and to explore disease pathogenesis. To investigate whether a similar pattern could be found also in blood samples, an immunoprecipitation (IP) based method for enrichment of Aß peptides from human plasma was developed. The peptides were analyzed using matrix-assisted-laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry for Aß profiling and selected reaction monitoring (SRM) for MS quantification of Aß1-38, Aß1-40 and Aß1-42 using tripe quadrupole MS. Sixteen N- or C-terminally truncated Aß peptides were reproducibly detected in human plasma, of which 11 were verified by tandem MS. In a pilot study including 9 AD patients and 10 controls, where Aß1-38, Aß1-40 and Aß1-42 were quantified using SRM, no AD-associated change in plasma levels of the peptides were observed. Using MS-based measurement techniques, we show that several Aß peptides can be monitored in a single analysis and the developed methods have the potential to be used as a read out in clinical trials of drugs affecting APP processing or Aß homeostasis.

Soluble amyloid precursor protein α and ß in CSF in Alzheimer's disease.

OBJECTIVE: Cerebral accumulation of amyloid ß (Aß) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by α- or ß-secretase results in two soluble metabolites, sAPPα and sAPPß, respectively. However, previous data have shown that both α- and ß-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPPα and sAPPß in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. METHODS: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPPα and sAPPß from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPPα. RESULTS: Four different C-terminal forms of sAPP were identified, sAPPß-M671, sAPPß-Y681, sAPPα-Q686, and sAPPα-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R(2)) between the two immunoassays was 0.41 for sAPPα and 0.45 for sAPPß. CONCLUSION: Using high resolution MS, we show here for the first time that sAPPα in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPPα and sAPPß levels are unaltered in AD.

An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid ß and amyloid precursor protein in human and cat cerebrospinal fluid.

Amyloid precursor protein (APP) is the precursor protein to amyloid ß (Aß), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aß peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aß peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aß peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aß processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aß was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aß (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aß peptide species in CSF by using an online top-down MS-based method.

Identification of novel α-synuclein isoforms in human brain tissue by using an online nanoLC-ESI-FTICR-MS method.

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn1₋140) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn1₋139 and Ac-α-syn1₋103) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).

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.

Proteomics and peptidomics in neuroscience. Experience of capabilities and limitations in a neurochemical laboratory.

The increasing use of proteomics has created a basis for new strategies to develop methodologies for rapid identification of protein patterns in living organisms. It has also become evident that proteomics has other potential applications than protein and peptide identification, e.g. protein characterization, with the aim of revealing their structure, function(s) and interactions of proteins. In comparative proteomics studies, the protein expression of a certain biological system is compared with another system or the same system under perturbed conditions. Global identification of proteins in neuroscience is extremely complex, owing to the limited availability of biological material and very low concentrations of the molecules. Moreover, in addition to proteins, there are number of peptides that must also be considered in global studies on the central nervous system. In this overview, we focus on and discuss problems related to the different sources of biological material and sample handling, which are part of all preparatory and analytical steps. Straightforward protocols are desirable to avoid excessive purification steps, since loss of material at each step is inevitable. We would like to merge the two worlds of proteomics/peptidomics and neuroscience, and finally we consider different practical and technical aspects, illustrated with examples from our laboratory.

Neurodevelopmental influences on the immune system reflecting brain pathology.

A number of studies have shown that early life events can affect the development of the nervous system, contributing to particular individual differences in later vulnerability to different forms of psychosocial stress related to the environment and lifestyle. Neuropeptides, chemokines (CKs), neurotrophins (NTs) belong to the chemical microenvironment of the cells of the central nervous system (CNS). This paper reviews research performed in our and other laboratories indicating that mass spectrometry should play a significant role in future studies of the structures of proteins/peptides in neuroscience. These applications include peptide metabolism associated with normal and impaired neurone/immune function. Detailed information about peptide/protein processing in the CNS may be studied by using the lymphocyte as a model reflecting different chemical modifications of peptides/proteins related to various psychosomatic disturbances reflecting disorders of environment and lifestyle.