Hillevi Englund

Sensitive ELISA detection of amyloid-β protofibrils in biological samples

Amyloid‐β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer’s disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic‐Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.

Amyloid-β oligomers are inefficiently measured by enzyme-linked immunosorbent assay

Amyloid‐β (Aβ) peptide levels are widely measured by enzyme‐linked immunosorbent assay (ELISA) in Alzheimers disease research. Here, we show that oligomerization of Aβ results in underestimated Aβ ELISA levels. The implications are that comprehensive analysis of soluble Aβ requires either sample pretreatment at denaturing conditions or novel conformation‐dependent immunoassays. Our findings might be of relevance for many neurodegenerative disorders in which soluble protein aggregates are the main neurotoxic species. Ann Neurol 2005;58:147–150

Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice

Oligomeric assemblies of amyloid‐β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long‐term potentiation. The tg‐ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg‐ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg‐ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg‐ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg‐ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg‐ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age‐dependent manner in tg‐ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.

An amyloid-β protofibril-selective antibody prevents amyloid formation in a mouse model of Alzheimer's disease

Human genetics link Alzheimers disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimers disease brain.

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

Mutations within the amyloid‐β (Aβ) 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 Aβ protofibrils in vitro and intraneuronal Aβ 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 β‐secretase cleavage, as demonstrated by altered levels of N‐ and C‐terminal APP fragments. Although the Arctic mutation is located close to the α‐secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease‐10, a major α‐secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for α‐secretase cleavage. As a result, the extent and subcellular location of Aβ formation is changed, as revealed by increased Aβ 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‐Aβ mutations, could relate to altered APP processing with increased steady‐state levels of Arctic Aβ, particularly at intracellular locations.

Oligomerization Partially Explains the Lowering of Aβ42 in Alzheimer’s Disease Cerebrospinal Fluid

Background/Objective: The lowering of natively analyzed Aβ42 in cerebrospinal fluid (CSF) is used as a diagnostic tool in Alzheimer’s disease (AD). The presence of Aβ oligomers can interfere with such analyses causing underestimation of Aβ levels due to epitope masking. The aim was to investigate if the lowering of CSF Aβ42 seen in AD is caused by oligomerization. Methods: Aβ42 was analyzed under both denaturing and non-denaturing conditions. An Aβ42 oligomer ratio was calculated from these quantifications. The presence of oligomers leads to Aβ42 epitope masking during non-denaturing assays, resulting in a higher ratio. Results: The Aβ42 oligomer ratio was used for the assessment of oligomerized Aβ in human CSF, after being evaluated in transgenic mouse brain homogenates. AD and mild cognitive impairment (MCI) samples displayed the expected decrease in natively measured Aβ42 compared to healthy controls and frontotemporal dementia, but not when analyzing under denaturing conditions. Accordingly, AD and MCI CSF had a higher Aβ42 oligomer ratio in CSF. Conclusion: Combining denaturing and non-denaturing quantifications of Aβ42 into an oligomer ratio enables the assessment of Aβ oligomers in biological samples. The increased Aβ42 oligomer ratio for AD and MCI indicates the presence of oligomers in CSF and that the lowering of natively measured Aβ42 is caused by oligomerization.

Interference from Heterophilic Antibodies in Amyloid-β Oligomer ELISAs

Amyloid-β (Aβ) oligomers of different sizes and forms have recently been the focus formany Alzheimers disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Aβ species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Aβ oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples.

Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: What have we learnt about functional glutamatergic neurotransmission?

Abstract During the past decade, three proteins that possess the capability of packaging glutamate into presynaptic vesicles have been identified and characterized. These three vesicular glutamate transporters, VGLUT1–3, are encoded by solute carrier genes Slc17a6–8. VGLUT1 (Slc17a7) and VGLUT2 (Slc17a6) are expressed in glutamatergic neurons, while VGLUT3 (Slc17a8) is expressed in neurons classically defined by their use of another transmitter, such as acetylcholine and serotonin. As glutamate is both a ubiquitous amino acid and the most abundant neurotransmitter in the adult central nervous system, the discovery of the VGLUTs made it possible for the first time to identify and specifically target glutamatergic neurons. By molecular cloning techniques, different VGLUT isoforms have been genetically targeted in mice, creating models with alterations in their glutamatergic signalling. Glutamate signalling is essential for life, and its excitatory function is involved in almost every neuronal circuit. The importance of glutamatergic signalling was very obvious when studying full knockout models of both VGLUT1 and VGLUT2, none of which were compatible with normal life. While VGLUT1 full knockout mice die after weaning, VGLUT2 full knockout mice die immediately after birth. Many neurological diseases have been associated with altered glutamatergic signalling in different brain regions, which is why conditional knockout mice with abolished VGLUT-mediated signalling only in specific circuits may prove helpful in understanding molecular mechanisms behind such pathologies. We review the recent studies in which mouse genetics have been used to characterize the functional role of VGLUT2 in the central nervous system.

Large Aggregates Are the Major Soluble Aβ Species in AD Brain Fractionated with Density Gradient Ultracentrifugation

Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimers Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.

Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

BackgroundProtein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimers disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.ResultsFor specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.ConclusionsThe proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimers disease and other neurodegenerative disorders.