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Alzheimer’s disease (AD) is the most common form of dementia and apart from the individual suffering AD also causes a large economic burden for society. AD is associated with progressive neurodegeneration and atrophy of the brain. Extracellular fibrillar assemblies of the amyloid-β peptide (Aβ) in the brain represent a clinical hallmark of AD and these are today considered to be the initial cause of the disease.  The tissue-damaging properties of Aβ assemblies are, however, linked to their structures. Aβ represents a spectrum of peptides between 38-43 residues that can adopt several structures that differ both concerning their morphology and pathological properties. The mechanisms by which Aβ self-assembles, the binding strength of these structures to Aβ monomers, as well as the cross-interaction between different Aβ variants are today not fully understood. Aβ amyloid formation follows a nucleation-dependent mechanism which implies that a kinetically unfavorable nucleus must form before the formation of an amyloid fibril. The elongation of the fibril then proceeds via a template-dependent mechanism where monomeric peptides are incorporated in a highly ordered manner. Using SPR the template-dependent mode of elongation can be selectively monitored. Here, we have used the technique to probe the binding strength of Aβ fibrils and in paper 1 the role of pH and the intrinsic histidines in the Aβ sequence were investigated. The result shows that the histidines do not contribute to the previously observed increase in fibrillar strength at low pH. In paper 2 we analyzed the cross-templation between the in vivo most common variants of Aβ, represented by Aβ1-40 and Aβ1-42. Within this work, we revealed two intrinsic mechanisms preventing Aβ to adopt the structure of the significantly more pathogenic Aβ1-42 variant. In paper 3 we characterized the effect of apolipoprotein E (ApoE) on Aβ amyloid formation. ApoE is today the strongest genetic linker to the development of AD and a well-known binding partner to Aβ fibrils in vivo. Using SPR we can here show that ApoE can prevent Aβ fibril elongation. Although ApoE effectively impairs fibril formation, preventing elongation may result in alternative assemblies with higher cytotoxic properties which hence may explain its pathological effect. In paper 4 we have linked SPR to scanning electron microscopy (SEM). The work presents a novel and generic approach to simultaneously monitor the kinetic properties of amyloid formation, the binding of ligands, and its morphology. We have here specifically probed the binding properties of ApoE to Aβ fibrils, and in combination with immunogold staining technique revealed its binding pattern. Taken together this work pioneers the use of SPR as a powerful technique to elucidate Aβ amyloid formation and the complex enigma of factors causing AD. 

Alzheimer's disease (AD) is strongly linked to amyloid depositions of the Aβ peptide (Aβ). The lipid-binding protein apolipoprotein E (ApoE) has been found to interfere with Aβ amyloid formation and to exert a strong clinical impact to the pathology of AD. The APOE gene exists in three allelic isoforms represented by APOE ε2, APOE ε3, and APOE ε4. Carriers of the APOE ε4 variant display a gene dose-dependent increased risk of developing the disease. Aβ amyloids are formed via a nucleation-dependent mechanism where free monomers are added onto a nucleus in a template-dependent manner. Using a combination of surface plasmon resonance and thioflavin-T assays, we here show that ApoE can target the process of fibril elongation and that its interference effectively prevents amyloid maturation. We expose a complex equilibrium where the concentration of ApoE, Aβ monomers, and the amount of already formed Aβ fibrils will affect the relative proportion and formation rate of mature amyloids versus alternative assemblies. The result illustrates a mechanism which may affect both the clearance rate of Aβ assemblies in vivo and the population of cytotoxic Aβ assemblies.

Apolipoprotein E (ApoE) has become a primary focus of research after the discovery of its strong linkage to Alzheimer’s disease (AD), where the ApoE4 variant is the highest genetic risk factor for this disease. ApoE is commonly found in amyloid deposits of different origins, and its interaction with amyloid-β peptide (Aβ), the hallmark of AD, is well known. However, studies on the interaction of ApoEs with other amyloid-forming proteins are limited. Islet amyloid polypeptide (IAPP) is an amyloid-forming peptide linked to the development of type-2 diabetes and has also been shown to be involved in AD pathology and vascular dementia. Here we studied the impact of ApoE on IAPP aggregation and IAPP-induced toxicity on blood vessel pericytes. Using both in vitro and cell-based assays, we show that ApoE efficiently inhibits the amyloid formation of IAPP at highly substoichiometric ratios and that it interferes with both nucleation and elongation. We also show that ApoE protects the pericytes against IAPP-induced toxicity, however, the ApoE4 variant displays the weakest protective potential. Taken together, our results suggest that ApoE has a generic amyloid-interfering property and can be protective against amyloid-induced cytotoxicity, but there is a loss of function for the ApoE4 variant.

Background: Immunogold labeling in combination with transmission electron microscopy analysis is a technique frequently used to correlate high-resolution morphology studies with detailed information regarding localization of specific antigens. Although powerful, the methodology has limitations and it is frequently difficult to acquire a stringent system where unspecific low-affinity interactions are removed prior to analysis.

Results: We here describe a combinatorial strategy where surface plasmon resonance and immunogold labeling are used followed by a direct analysis of the sensor-chip surface by scanning electron microscopy. Using this approach, we have probed the interaction between amyloid-beta fibrils, associated to Alzheimer's disease, and apolipoprotein E, a well-known ligand frequently found co-deposited to the fibrillar form of A beta in vivo. The results display a lateral binding of ApoE along the amyloid fibrils and illustrates how the gold-beads represent a good reporter of the binding.

Conclusions: This approach exposes a technique with generic features which enables both a quantitative and a morphological evaluation of a ligand-receptor based system. The methodology mediates an advantage compared to traditional immunogold labeling since all washing steps can be monitored and where a high stringency can be maintained throughout the experiment.

We demonstrate the use of Scanning Electron microscopy (SEM) in combination with Surface Plasmon Resonance (SPR) to probe and verify the formation of amyloid and its morphology on an SPR chip. SPR is a technique that measures changes in the immobilized weight on the chip surface and is frequently used to probe the formation and biophysical properties of amyloid structures. In this context it is of interest to also monitor the morphology of the formed structures. The SPR chip surface is made of a layer of gold, which represent a suitable material for direct analysis of the surface using SEM. The standard SPR chip used here (CM5-chip, GE Healthcare, Uppsala, Sweden) can easily be disassembled and directly analyzed by SEM. In order to verify the formation of amyloid fibrils in our experimental conditions we analyzed also in-solution produced structures by using Transmission Electron Microscopy (TEM). For further details and experimental findings, please refer to the article published in Journal of Molecular Biology, (Brännström K. et al., 2018) [1].

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1-40 and Aβ1-42 are the dominant forms. The fibril architectures of Aβ1-40 and Aβ1-42 differ and Aβ1-42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1-42 can be cross-templated and incorporated into the ends of Aβ1-40 fibrils, while incorporation of Aβ1-40 monomers into Aβ1-42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1-40 to incorporate into the ends of Aβ1-42 fibrils and the capacity of Aβ1-42 monomers to adopt the properties of Aβ1-40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1-42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1-40 from adopting the fibrillar properties of Aβ1-42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.

Low pH has a strong stabilising effect on the fibrillar assembly of amyloid β, which is associated with Alzheimer's disease. The stabilising effect is already pronounced at pH 6.0, suggesting that protonation of histidines might mediate this effect. Through the systematic substitution of the three native histidines in Aβ for alanines, we have evaluated their role in fibril stability. Using surface plasmon resonance, we show that at neutral pH the fibrillar forms of all His-Ala variants are destabilised by a factor of 4-12 compared to wild-type Aβ. However, none of the His-Ala Aβ variants impair the stabilising effect of the fibril at low pH.