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.

ß-site amyloid precursor protein-cleaving enzyme 1(BACE1) inhibitor treatment induces Aß5-X peptides through alternative amyloid precursor protein cleavage.

INTRODUCTION: The ß-secretase enzyme, ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1), cleaves amyloid precursor protein (APP) in the first step in ß-amyloid (Aß) peptide production. Thus, BACE1 is a key target for candidate disease-modifying treatment of Alzheimer's disease. In a previous exploratory Aß biomarker study, we found that BACE1 inhibitor treatment resulted in decreased levels of Aß1-34 together with increased Aß5-40, suggesting that these Aß species may be novel pharmacodynamic biomarkers in clinical trials. We have now examined whether the same holds true in humans. METHODS: In an investigator-blind, placebo-controlled and randomized study, healthy subjects (n =18) were randomly assigned to receive a single dose of 30 mg of LY2811376 (n =6), 90 mg of LY2811376 (n =6), or placebo (n =6). We used hybrid immunoaffinity-mass spectrometry (HI-MS) and enzyme-linked immunosorbent assays to monitor a variety of Aß peptides. RESULTS: Here, we demonstrate dose-dependent changes in cerebrospinal fluid (CSF) Aß1-34, Aß5-40 and Aß5-X after treatment with the BACE1-inhibitor LY2811376. Aß5-40 and Aß5-X increased dose-dependently, as reflected by two independent methods, while Aß1-34 dose-dependently decreased. CONCLUSION: Using HI-MS for the first time in a study where subjects have been treated with a BACE inhibitor, we confirm that CSF Aß1-34 may be useful in clinical trials on BACE1 inhibitors to monitor target engagement. Since it is less hydrophobic than longer Aß species, it is less susceptible to preanalytical confounding factors and may thus be a more stable marker. By independent measurement techniques, we also show that BACE1 inhibition in humans is associated with APP-processing into N-terminally truncated Aß peptides via a BACE1-independent pathway. TRIAL REGISTRATION: ClinicalTrials.gov NCT00838084. Registered: First received: January 23, 2009, Last updated: July 14, 2009, Last verified: July 2009.

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.

Altered cerebrospinal fluid levels of amyloid ß and amyloid precursor-like protein 1 peptides in Down's syndrome.

Down's syndrome (DS) patients develop early Alzheimer's disease pathology with abundant cortical amyloid plaques, likely due to overproduction of the amyloid precursor protein (APP), which subsequently leads to amyloid ß (Aß) aggregation. This is reflected in cerebrospinal fluid (CSF) levels of the 42-amino acid long Aß peptide (Aß1-42), which are increased in young DS patients and decreases with age. However, it is unclear whether DS also affects other aspects of Aß metabolism, including production of shorter C- and N-terminal truncated Aß peptides, and production of peptides from the amyloid precursor-like protein 1 (APLP1), which is related to APP, and cleaved by the same enzymatic processing machinery. APLP1-derived peptides may be surrogate markers for Aß1-42 production in the brain. Here, we used hybrid immunoaffinity-mass spectrometry and enzyme-linked immunosorbent assays to monitor several Aß and APLP1 peptides in CSF from DS patients (n = 12) and healthy controls (n = 20). CSF levels of Aß1-42 and three endogenous peptides derived from APLP1 (APL1ß25, APL1ß27 and APL1ß28) were decreased in DS compared with controls, while a specific Aß peptide, Aß1-28, was increased in a majority of the DS individuals. This study indicates that DS causes previously unknown specific alterations of APP and APLP1 metabolism.