Elevated soluble amyloid beta protofibrils in Down syndrome and Alzheimer's disease.

Down syndrome (DS) is caused by trisomy of chromosome 21, which leads to a propensity to develop amyloid ß (Aß) brain pathology in early adulthood followed later by cognitive and behavioral deterioration. Characterization of the Aß pathology is important to better understand the clinical deterioration of DS individuals and to identify interventive strategies. Brain samples from people with DS and Alzheimer's disease (AD), as well as non-demented controls (NDC), were analyzed with respect to different Aß species. Immunohistochemical staining using antibodies towards Aß was also performed. Elevated levels of soluble Aß protofibrils and insoluble Aßx-40 and Aßx-42 in formic acid brain extracts, and elevated immunohistochemical staining of Aß deposits were demonstrated with the antibody BAN2401 (lecanemab) in DS and AD compared with NDC. These data and the promising data in a large phase 2 CE clinical trial with lecanemab suggest that lecanemab may have the potential to preserve cognitive capacity in DS. Lecanemab is currently in a phase 3 CE clinical trial.

Characterization of monomeric and soluble aggregated Aß in Down's syndrome and Alzheimer's disease brains.

The major characteristics of Alzheimer's disease (AD) are amyloid plaques, consisting of aggregated beta amyloid (Aß) peptides, together with tau pathology (tangles, neuropil treads and dystrophic neurites surrounding the plaques), in the brain. Down's syndrome (DS) individuals are at increased risk to develop AD-type pathology; most DS individuals have developed substantial pathology already at the age of 40. DS individuals have an extra copy of chromosome 21, harbouring the amyloid precursor protein gene (APP). Our aim was to investigate the Aß peptide pattern in DS and AD brains to investigate differences in their amyloid deposition and aggregation, respectively. Cortical tissue from patients with DS (with amyloid pathology), sporadic AD and controls were homogenized and fractionated into TBS (water soluble) and formic acid (water insoluble) fractions. Immunoprecipitation (IP) was performed using a variety of antibodies targeting different Aß species including oligomeric Aß. Mass spectrometry was then used to evaluate the presence of Aß species in the different patient groups. A large number of Aß peptides were identified including Aß1-X, 2-X, 3-X, 4-X, 5-X, 11-X, and Aß peptides extended N terminally of the BACE1 cleavage site and ending at amino 15 in the Aß sequence APP/Aß(-X to 15), as well as peptides post-translationally modified by pyroglutamate formation. Most Aß peptides had higher abundance in AD and DS compared to controls, except the APP/Aß(-X to 15) peptides which were most abundant in DS followed by controls and AD. Furthermore, the abundancies of AßX-40 and AßX-34 were increased in DS compared with AD. Aß1-40, Aß1-42, and Aß4-42 were identified as the main constitutes of protofibrils (IP'd using mAb158) and higher relative Aß1-42 signals were obtained compared with samples IP'd with 6E10 + 4G8, indicating that the protofibrils/oligomers were enriched with peptides ending at amino acid 42. All Aß peptides found in AD were also present in DS indicating similar pathways of Aß peptide production, degradation and accumulation, except for APP/Aß(-X to 15). Likewise, the Aß peptides forming protofibrils/oligomers in both AD and DS were similar, implying the possibility that treatment with clinical benefit in sporadic AD might also be beneficial for subjects with DS.

The murine version of BAN2401 (mAb158) selectively reduces amyloid-ß protofibrils in brain and cerebrospinal fluid of tg-ArcSwe mice.

Amyloid-ß (Aß) immunotherapy for Alzheimer's disease (AD) has good preclinical support from transgenic mouse models and clinical data suggesting that a long-term treatment effect is possible. Soluble Aß protofibrils have been shown to exhibit neurotoxicity in vitro and in vivo, and constitute an attractive target for immunotherapy. Here, we demonstrate that the humanized antibody BAN2401 and its murine version mAb158 exhibit a strong binding preference for Aß protofibrils over Aß monomers. Further, we confirm the presence of the target by showing that both antibodies efficiently immunoprecipitate soluble Aß aggregates in human AD brain extracts. mAb158 reached the brain and reduced the brain protofibril levels by 42% in an exposure-dependent manner both after long-term and short-term treatment in tg-ArcSwe mice. Notably, a 53% reduction of protofibrils/oligomers in cerebrospinal fluid (CSF) that correlated with reduced brain protofibril levels was observed after long-term treatment, suggesting that CSF protofibrils/oligomers could be used as a potential biomarker. No change in native monomeric Aß42 could be observed in brain TBS extracts after mAb158-treatment in tg-ArcSwe mice. By confirming the specific ability of mAb158 to selectively bind and reduce soluble Aß protofibrils, with minimal binding to Aß monomers, we provide further support in favor of its position as an attractive new candidate for AD immunotherapy. BAN2401 has undergone full phase 1 development, and available data indicate a favorable safety profile in AD patients.

Off-pathway α-synuclein oligomers seem to alter α-synuclein turnover in a cell model but lack seeding capability in vivo.

Aggregated α-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Experimental evidence indicates that α-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of α-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when α-synuclein oligomers were added to monomeric α-synuclein. In contrast, exogenously added α-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing α-synuclein that were treated with the oligomers displayed reduced α-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected α-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of α-synuclein oligomers into the neocortex of α-synuclein transgenic mice did not induce formation of proteinase K resistant α-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced α-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.

Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α-synuclein transgenic mice with the disease-causing A30P mutation.

Inclusions of intraneuronal alpha-synuclein (α-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α-synuclein is a central feature of the disease pathogenesis. Among the different α-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α-synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α-synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α-synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α-synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective α-synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.

Antibodies against alpha-synuclein reduce oligomerization in living cells.

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.

Docosahexaenoic acid stimulates non-amyloidogenic APP processing resulting in reduced Abeta levels in cellular models of Alzheimer's disease.

Epidemiological studies suggest that a high intake of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), is associated with a reduced risk of Alzheimer's disease. Here, we examined the effects of DHA on amyloid precursor protein (APP) processing in cellular models of Alzheimer's disease by analysing levels of different APP fragments, including amyloid-beta (Abeta). DHA administration stimulated non-amyloidogenic APP processing and reduced levels of Abeta, providing a mechanism for the reported beneficial effects of DHA in vivo. However, an increased level of APP intracellular domain was also observed, highlighting the need to increase our knowledge about the relevance of this fragment in Alzheimer's disease pathogenesis. In conclusion, our results suggest that the proposed protective role of DHA in Alzheimer's disease pathogenesis might be mediated by altered APP processing and Abeta production.

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase.

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.

Assessing Secchi and photic zone depth in the Baltic Sea from satellite data.

Long-term trends in the Secchi depth of the Baltic Sea have been interpreted in terms of eutrophication. The spectral attenuation coefficient Kd (490) can be estimated from remote sensing data. Given the empirical and theoretical relationships between diffuse attenuation and Secchi depth, it is therefore possible to estimate the trophic state from remote sensing data. This paper considers relationships among remotely sensed and in-water measured K (490), and Secchi depth data obtained during dedicated sea-truthing campaigns in the eastern Baltic Proper in 1999 (4) and in the western Baltic Proper/Himmerfjärden area during 2001 and 2002. In-water measurements are used to establish the relationship between the PAR and the spectral attenuation coefficient in the Baltic Sea via regression analysis. The analysis showed that in the area of investigation Kd(490) is about 1.48 times higher than Kd (PAR). This relationship is then used to define the link between the photic zone depth and the remote sensing optical depth, Kd (490)-1. The results show that the depth of the euphotic zone is about 6.8 times Kd (490)-1. The regression analysis between Kd (PAR) and Secchi depth confirmed previous work that Kd (PAR) is about 1.7 of the inverse Secchi depth. Furthermore, an in-water algorithm between Secchi depth and Kd (490) is used to simulate a Secchi depth map of the Baltic Sea from SeaWiFS Kd(490) data. This map is verified against sea-truthing data. Kd (490) data derived from satellite is compared to in situ Kd (490), and the sources of error are discussed.