Reduced Influence of apoE on Aß43 Aggregation and Reduced Vascular Aß43 Toxicity as Compared with Aß40 and Aß42.

The amyloid-ß 43 (Aß43) peptide has been shown to be abundantly expressed in Alzheimer's disease plaques, whereas only relatively low levels have been demonstrated in cerebral amyloid angiopathy (CAA). To better understand this discrepant distribution, we studied various biochemical properties of Aß43, in comparison with Aß40 and Aß42. We assessed the interaction of Aß43 with the three apoE isoforms (apoE2, apoE3, and apoE4) using SDS-PAGE/Western blotting and ELISA, aggregation propensity using thioflavin T assays, and cytotoxicity towards cerebrovascular cells using MTT assays. We found that Aß43 did not differ from Aß42 in its interaction with apoE, whereas Aß40 had a significantly lower degree of interaction with apoE. At a molar ratio of 1:100 (apoE:Aß), all apoE isoforms were comparably capable of inhibiting aggregation of Aß40 and Aß42, but not Aß43. All Aß variants had a concentration-dependent negative effect on metabolic activity of cerebrovascular cells. However, the degree of this effect differed for the three Aß isoforms (Aß40 > Aß42 > Aß43), with Aß43 being the least cytotoxic peptide towards cerebrovascular cells. We conclude that Aß43 has different biochemical characteristics compared with Aß40 and Aß42. Aggregation of Aß43 is not inhibited by apoE, in contrast to the aggregation of Aß40 and Aß42. Furthermore, cerebrovascular cells are less sensitive towards Aß43, compared with Aß40 and Aß42. In contrast, Aß43 neither differed from Aß42 in its aggregation propensity (in the absence of apoE) nor in its apoE-binding capacity. Altogether, our findings may provide an explanation for the lower levels of Aß43 accumulation in cerebral vessel walls.

Limitations of the hCMEC/D3 cell line as a model for Aß clearance by the human blood-brain barrier.

Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid-ß (Aß) at the cerebrovasculature due to decreased clearance at the blood-brain barrier (BBB). However, the exact mechanism of Aß clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Aß clearance across the BBB, with an emphasis on brain-to-blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Aß was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Aß. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Aß clearance. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

CSF d-serine concentrations are similar in Alzheimer's disease, other dementias, and elderly controls.

Cerebrospinal fluid (CSF) levels of d-serine were recently reported as a potential new biomarker for Alzheimer's disease (AD), showing a perfect distinction between AD patients and healthy controls. In this study, we aimed to confirm these results and extend these previous findings to dementia with Lewy bodies and frontotemporal dementia. d-Serine levels in CSF of 29 AD patients, 8 dementia with Lewy bodies patients, 14 frontotemporal dementia patients, and 28 nondemented controls were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. In contrast to previous findings, in our study CSF d-serine levels were only slightly increased in AD patients compared with controls. CSF d-serine in AD did not differ from other dementias and was also not correlated to mini-mental state examination-scores. Owing to the large overlap of d-serine levels, we conclude that CSF d-serine is neither a suitable biomarker for AD nor for cognitive decline.

Amyloid-ß oligomer detection by ELISA in cerebrospinal fluid and brain tissue.

Amyloid-ß (Aß) deposits are important pathological hallmarks of Alzheimer's disease (AD). Aß aggregates into fibrils; however, the intermediate oligomers are believed to be the most neurotoxic species and, therefore, are of great interest as potential biomarkers. Here, we have developed an enzyme-linked immunosorbent assay (ELISA) specific for Aß oligomers by using the same capture and (labeled) detection antibody. The ELISA predominantly recognizes relatively small oligomers (10-25 kDa) and not monomers. In brain tissue of APP/PS1 transgenic mice, we found that Aß oligomer levels increase with age. However, for measurements in human samples, pretreatment to remove human anti-mouse antibodies (HAMAs) was required. In HAMA-depleted human hippocampal extracts, the Aß oligomer concentration was significantly increased in AD compared with nondemented controls. Aß oligomer levels could also be quantified in pretreated cerebrospinal fluid (CSF) samples; however, no difference was detected between AD and control groups. Our data suggest that levels of small oligomers might not be suitable as biomarkers for AD. In addition, we demonstrate the importance of avoiding HAMA interference in assays to quantify Aß oligomers in human body fluids.