Binding of ApoE isoforms to Aβ peptides and effects on their fibrillization

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T. S. Wärmländer, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8086961/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alzheimer’s disease (AD) is the most widespread neurodegenerative disease, strongly linked to amyloid depositions in the brain consisting of amyloid beta (Aβ) peptides. The likelihood of developing late-onset Alzheimer’s disease (LOAD) is influenced by the specific isoforms of apolipoprotein E (ApoE), with ApoE4 being the strongest known genetic risk factor for LOAD. Strong evidence suggests that ApoE impacts AD by modulating Aβ aggregation and clearance, although the precise molecular mechanisms remain incompletely understood. Microscale thermophoresis (MST) is a powerful technique for characterizing molecular interactions in solution, which has been used to determine various binding constants, although not the binding of ApoE to Aβ peptides. MST results show that ApoE isoforms bind Aβ1–40 and Aβ1–42 with low micromolar affinity. For Aβ1–42, ApoE3 shows the strongest binding (K d = 0.72 µM) and ApoE4 the weakest (K d = 2.80 µM). For Aβ1–40, ApoE4 shows the strongest binding (K d = 1.59 µM) and ApoE2 the weakest (K d = 5.29 µM). The MST results show that ApoE interacts with Aβ peptides at supraphysiological peptide concentrations. However, ApoE inhibited the fibrillization of Aβ1–40 peptide at sub-stoichiometric concentrations, which might be related to blocking Aβ fibril elongation in vivo. The estimated IC 50 values indicate that ApoE4 has slightly stronger and ApoE2 has the lowest inhibitory effect on Aβ1–42 fibrillization. Biophysics Alzheimer’s disease amyloid β peptide apolipoprotein E microscale thermophoresis fibrillization ThT assay Figures Figure 1 Figure 2 Introduction Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, characterized by the occurrence of amyloid deposits in the brain composed of Aβ (amyloid-beta) peptides. The risk of developing late-onset Alzheimer’s disease (LOAD) is dependent on the isoforms of apolipoprotein E (ApoE) that individuals inherit. ApoE exists primarily in three isoforms: ApoE2 (Cys112, Cys158), ApoE3 (Cys112, Arg158), and ApoE4 (Arg112, Arg158) 1 . ApoE is co-localized with Aβ in senile plaques of AD and has been shown to bind tightly to immobilized Aβ peptide 2 . Notably, the presence of ApoE4 is recognized as the highest genetic risk factor for developing LOAD 2 . There is substantial evidence that ApoE influences AD by affecting Aβ aggregation and clearance 3 ; however, the molecular mechanism underlying this remains poorly understood. The direct interaction of ApoE with Aβ peptides has been studied mostly by ELISA; however, a few reports have used dual polarization interferometry (DPI) and surface plasmon resonance. According to ELISA, the dissociation constant values for ApoE-Aβ complexes are 48 nM (ApoE2), 63.7 nM (ApoE3), and 75.9 nM (ApoE4) (Yamauchi et al. 1999). Similar study using cell-derived ApoE (Sf9, HEK, and RAW cells) resulted in the following values: 13.3 nM (ApoE3), 13.9 nM (ApoE4) for Aβ1–42 and 9.3 nM (ApoE3), 10.3 nM (ApoE4) for Aβ1–40 (Tokuda et al. 2000). According to Sadowski et al., the dissociation constant of Aβ1–40 complex with lipidated ApoE4 determined by ELISA is 14.7 nM (Sadowski et al. 2006). One study using DPI determined K d values for ApoE and Aβ1–40 to be 251 nM (ApoE2), 40 nM (ApoE3), and 24.6 nM (ApoE4) 4 . Surface plasmon resonance was used by Rahman et al. to determine K d values for ApoE and Aβ1–42 protofibrils to be 3 nM 5 . Liu et al. used ELISA to map the binding regions between ApoE and Aβ, identifying residues 244–272 on ApoE and 12–28 on the Aβ peptide as the interacting sites 6 . Using a different approach, MD simulations, it was proposed that Aβ residues Asp1 and Asp23 might interact with cationic residues of ApoE, and thereby perturb its salt bridge network, especially in ApoE4 7 . The effect of ApoE on fibrillization of Aβ has been widely studied; however, the results are conflicting. By some authors, it has been found that ApoE has a direct role in promoting and accelerating fibril formation of Aβ1–40, where ApoE4 was more efficient than ApoE3 at enhancing amyloid formation 8 . Sadowski et al. also showed that adding lipidated human ApoE4 significantly increased the amount of Aβ1–40 fibrils 9 . Lipidated ApoE has been shown to increase Aβ oligomer levels in an isoform-dependent manner, with lipidated ApoE4 exerting the greatest effect on Aβ oligomer formation 10 . Liao et al. claimed that ApoE-mediated plaque formation may be the result of ApoE aggregation, as evidenced by their observation that anti-human ApoE4 antibody binds nonlipidated and aggregated ApoE4 from amyloid plaques in mice, reducing Aβ deposition 11 . On the contrary, Evans et al. showed that ApoE inhibits amyloid formation at substoichiometric levels 12 . Furthermore, Garai et al. reported that at low concentrations, ApoE binds to and stabilizes Aβ oligomers, whereas at higher concentrations, it interacts with Aβ fibrils, stabilizing them and thereby inhibiting further fibril formation 13 . In addition, ApoE has been shown to slow down the oligomerization of Aβ1–40 in the solution 14 . The findings of Ghosh et al. also support the inhibitory effect, as they show that the Aβ1–42 aggregation is delayed dramatically in the presence of stoichiometric concentrations of both lipid-free and lipidated ApoE forms, and that ApoE interacts rather with fibrillization intermediates than with the monomers of the Aβ peptide 15 . Recently, the same group reported that lipidated ApoE inhibits the elongation of Aβ1–42 fibrils in an isoform-dependent manner, where ApoE2 and ApoE3 exhibited the strongest inhibitory effects, while secondary nucleation was largely unaffected 16 . According to Islam et al., ApoE inhibits the process of fibril elongation and prevents amyloid maturation 17 . Xia et al. indicated that all ApoE isoforms associate with Aβ in the early stages of fibrillization and then fall away as fibrillization occurs 3 . It has also been shown that ApoE modulates the aggregation, clearance, and toxicity of Aβ in an isoform- and lipidation-specific way, by selectively removing non-lipidated ApoE4-Aβ co-aggregates and enhancing the clearance of toxic Aβ by glial cells 3 . Ghosh et al. found that both ApoE3 and ApoE4 also suppress at substoichiometric levels the aggregation of other proteins, for example, the AD-related proinflammatory protein S100A9, in a concentration-dependent manner 18 . MST is a powerful method for characterizing molecular interactions in solutions 19 . To date, MST has been used to study ApoE binding to complement regulator factor H 20 , various chemical probes 21 , and α-synuclein 22 . In this study, we utilized MST to examine the interaction between Aβ peptides and ApoE and determined the effect of ApoE proteins on the fibrillization of Aβ1–42 using the ThT assay. Our results indicate that Aβ peptides interact with ApoE at micromolar concentrations, whereas ApoE inhibits fibrillization of Aβ1–42 at substoichiometric concentrations. Results and Discussion Microscale thermophoresis (MST) The binding of Aβ peptides to ApoE isoforms was studied using MST with fluorescently labelled ApoE (20 nM) isoforms (E2/E3/E4) as the target and Aβ1–42 or Aβ1–40 as the ligand. The obtained K d values remained in the range of 1–2 µM (Fig. 1 , Tables 1 and 2 ) except that for ApoE2 binding to Aβ1–40 (Fig. 1 d, Table 2 ), characterized by the largest K d value, approx. 5 µM. The smallest K d value was below 1 µM and was observed for ApoE3 binding to Aβ1–42. Table 1 K d and RMSE values for binding of ApoE isoforms to Aβ1–42 (Fig. 1 , a, b, c). # (Aβ1–42) ApoE2 K d (µM) ± RMSE ApoE3 K d (µM) ± RMSE ApoE4 K d (µM) ± RMSE I 1.85 ± 0.13 0.76 ± 0.07 2.91 ± 0.10 II 1.87 ± 0.13 0.73 ± 0.16 2.70 ± 0.16 III 1.58 ± 0.09 0.68 ± 0.06 2.77 ± 0.11 Table 2 K d and RMSE values for binding of ApoE isoforms to Aβ1–40 (Fig. 1 , d, e, f). # (Aβ1–40) ApoE2 K d (µM) ± RMSE ApoE3 K d (µM) ± RMSE ApoE4 K d (µM) ± RMSE I 5.52 ± 0.04 2.07 ± 0.10 1.82 ± 0.28 II 5.02 ± 0.06 2.27 ± 0.07 1.44 ± 0.38 III 5.34 ± 0.08 1.74 ± 0.16 1.50 ± 0.23 Our results show that the K d values for ApoE-Aβ complexes determined by MST experiments show significantly weaker affinity than those estimated earlier by ELISA and DPI. The observed difference can be attributed to the nature of the measurements: MST measurements determine interactions in solutions, whereas ELISA and DPI evaluate the affinity of ApoE to the peptide immobilized on a solid surface. It is well known that affinity values vary depending on the experimental conditions and the technique used for the measurements 23 . The current MST results indicate that the interaction of ApoE with Aβ peptide occurs at supraphysiological micromolar concentrations, suggesting that ApoE interaction with Aβ peptides is unlikely to occur under physiological conditions. Measured K d values varied only slightly, with differences ranging from three to fourfold, depending on the type of ApoE or Aβ isoform. ApoE4 exhibits the weakest binding to Aβ1–42 but the strongest binding to Aβ1–40. The experiments were conducted using lipid-free ApoE, which can bind nonpolar substrates, and it could be suggested that in the case of lipidated ApoE, the K d values may be even higher. Our findings align with those of Verghese et al., who used multiple biochemical and analytical techniques to demonstrated minimal binding of lipidated ApoE to Aβ peptide in physiological fluids, with a slight increase in binding observed when the lipidation level was reduced 24 . Fluorescence spectrophotometry The IC 50 values for ApoE, determined from the inhibition of Aβ fibrillization, were lower than the K d values. Notably, the inhibition occurred already at low substoichiometric concentrations: 50 times less ApoE reduces the rate of Aβ1–42 fibrillization by 1.5 to 2.5 times. The inhibitory effect of ApoE on the fibrillization rate of Aβ1–42 may be explained by ApoE blocking of Aβ1–42 fibrillization sites. Recently, Dasadhikari et al. reported that the lipidated ApoE inhibits Aβ1–42 fibril elongation, while secondary nucleation remains largely unaffected 16 . Single fibril studies confirmed that the inhibition of the elongation rate is proportional to the binding of ApoE to the terminal ends of the fibrils. The affinity constants of ApoE isoforms for fibril ends were isoform-specific, with ApoE4 exhibiting fourfold weaker binding compared to ApoE2 and ApoE3 16 . In our study, we observed rather similar inhibitory effects of different ApoE isoforms on Aβ1–42 fibril elongation rate, whereas ApoE4 showed only slightly stronger inhibition. Fibrillization experiments, similarly to MST experiments, were performed with lipid-free ApoE, and therefore, the results may be different in the case of lipidated ApoE forms. Current results show that ApoE has a weak ability to interact with Aβ peptides; however, its ability to interact with intermediates of Aβ fibrillization is substantially higher. Therefore, ApoE can block the fibrillization of Aβ peptides, where the effects of different ApoE isoforms are quite similar. According to our results, the isoform-specific behavior of ApoE in the pathogenesis of AD is not linked to its interaction with Aβ monomers or aggregation intermediates. Still, it might be connected to its other interactions. The ApoE genotype may contribute to AD pathogenesis through several other distinct mechanisms. One hypothesis is related to LDL receptor-related protein 1 (LRP1), which is involved in the internalization and degradation of Aβ 25 26 . ApoE has been shown to disrupt Aβ clearance from the brain 27 , and blocking the ApoE/Aβ interactions resulted in enhanced Aβ clearance from the brain and decreased plaque deposition 9 , 28 . Therefore, ApoE isoforms may influence Aβ metabolism by competing for the same clearance pathways in the brain 24 . Alternatively, there might occur ApoE isoform-specific microglial lipid droplet accumulation, which leads to Tau phosphorylation and neurotoxicity in an ApoE-dependent manner 29 . It is also known that ApoE4 increases tau pathogenesis and leads to increased astroglial and microglial-mediated persistent inflammation, which leads to significant neurodegeneration in the presence of ApoE4 30 . The mystery of how slight modifications in the protein sequence of ApoE isoforms affect the propensity of AD remains to be established. Materials and Methods Lyophilized Aβ1–40 and Aβ1–42 were purchased from r-Peptide (Watkinsville, GA, USA); ApoE isoforms (E2/E3/E4) were purchased from AlexoTech AB (Umeå, Sweden). The integrity of the proteins were verified by MALDI-MS. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), HEPES, NaCl, NaOH, glycerol, NH 4 OH, and Thioflavin-T (ThT) were purchased from Sigma-Aldrich. Pluronic F-127, labelling buffer (LB-NHS), RED-NHS 2nd Generation dye, B-column, and DMSO were included in Protein Labelling Kit RED-NHS 2nd Generation (MO-L011) and purchased from Nanotemper (Nano Temper Technologies, München, Germany). Sample preparation Lyophilized Aβ1–40 and Aβ1–42 were solubilized in HFIP at a concentration of 100 µM, divided into aliquots, and the HFIP was evaporated under vacuum. The tubes with Aβ film were stored at -80°C. HFIP treatment is used to disassemble peptide aggregates into monomers. ApoE isoforms (E2/E3/E4) were dissolved in a 5 mM NaOH solution to a final concentration of 50 µM, divided into aliquots, and stored at -20°C. Microscale thermophoresis (MST) ApoE isoforms were diluted to a final concentration of 10 µM with labelling buffer (LB-NHS in MO-L011, Nanotemper), and labelled with NHS reactive dye (30 µM, dissolved in DMSO) from Protein Labelling Kit RED-NHS 2nd Generation (MO-L011; Nanotemper, München, Germany). The labelling reaction was carried out for 30 min at 25°C in the dark, the mixture was applied to the column according to the manufacturer’s instructions, and eluted with 50 mM HEPES, 150 mM NaCl, pH = 7.4, 10% glycerol, 0.1% Pluronic F-127. 10% glycerol was added to the buffer to decrease the aggregation of ApoE. Labelling efficiency was estimated using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) to be in the range of DOL = 0,5 − 1. Labelled ApoE isoforms were divided into aliquots and stored at -20°C. Before use, the labelled ApoE isoforms were thawed, diluted to 40 nM in 100 mM HEPES, 300 mM NaCl, pH 7.4, 20% glycerol, 0.2% Pluronic F-127, and centrifuged at 21,000 g, 4°C, for 10 min. HFIP-treated Aβ1–42 and Aβ1–40 aliquots were dissolved in 5 mM NaOH to a final concentration of 40 µM and 100 µM, respectively, incubated for 10 min on ice, and centrifuged for 10 min, 4°C, 21,000 g before use. Since amyloid β tends to aggregate at neutral pH, the 16-point serial dilutions of Aβ1–40 (100 µM) and Aβ1–42 (40 µM) were made in 5 mM NaOH. Samples for measurement were prepared by mixing 10 µl of a 16-point serial dilution of Aβ1–40 (100 µM) or Aβ1–42 (40 µM) with an equal amount of labelled ApoE isoforms (40 nM) with 10 µl of Aβ1–40 or Aβ1–42 to the final concentration of Aβ1–40 and Aβ1–42 ranging from 50 µM to 3 nM and 20 µM to 1,3 nM, respectively. Samples were measured in 50 mM HEPES, 150 mM NaCl, pH = 7.4, containing 10% glycerol and 0.1% Pluronic F-127. After mixing, the samples were instantly loaded into Standard capillaries, inserted into the Monolith NT 115 system (Nano Temper Technologies, München, Germany), and measured using 20% LED and low MST intensity (20% IR-laser power), with temperature control at 23°C. Results were analyzed with MO Affinity Analysis software and fitted by the K d equation provided by the software. MST-on time used for analysis was 20 seconds. The K d values, together with root mean square error (RMSE), were estimated using MO Affinity Analysis Software. Fluorescence spectrophotometry HFIP-treated Aβ1–42 was dissolved in 0.02% NH 4 OH, incubated 10 min on ice, and diluted with 40 mM HEPES, 200 mM NaCl, pH = 7.4 to a final concentration of 5 µM Aβ1–42, 20 mM HEPES, 100 mM NaCl, pH = 7.4. Fibrillization of Aβ1–42 was studied using fluorescent ligand ThT, whose fluorescence intensity at 480 nm (excitation at 440 nm) is increased upon binding to amyloid fibrils. ThT fluorescence was monitored on a Perkin Elmer (Waltham, MA, USA) LS55 fluorescence spectrophotometer in a 500 µL cuvette by constant stirring at 40°C in the presence of 5 µM ThT. To study the effect of ApoE isoforms on Aβ1–42 fibrillization, Aβ1–42 fibrillization curves in the absence and presence of different concentrations of ApoE2, ApoE3, and ApoE4 were determined and fitted to the Boltzmann Eq. ( 1 ) by the program Origin 8.5 (OriginLab Corporation, USA): $$\:\text{y}=\frac{{\text{A}}_{1}-{\text{A}}_{2}}{1+{e}^{\left(t-{t}_{0}\right)\bullet\:k}}\:+{\text{A}}_{2}$$ 1 where A1 is the initial fluorescence intensity level, A2 corresponds to the fluorescence at maximal fibrillization level, t0 is the time t when fluorescence is reached half maximum, and k is the apparent rate constant for the growth of fibrils. The half maximal inhibitory concentration (IC 50 ) values were calculated from the effects of ApoE isoforms on the apparent rate constant of fibril formation (IC 50 k ) according to hyperbolic dose-response curves: $$\:\text{y}={\text{A}}_{2}-\frac{{A}_{2}\text{*}\text{c}}{{\text{I}\text{C}}_{50}+\text{c}}$$ 2 where y is the fluorescence intensity of ThT, A2 is the maximum value of ThT fluorescence, c is the concentration of the test substance, and IC 50 is the concentration that reduces ThT fluorescence by 50%. Normalization of data and nonlinear regression analysis were carried out using the program Origin 8.5. Declarations Competing interest The authors declare no conflicts of interest. Author contributions Conceptualization: VT and PP; Investigation: MS acquired the Microscale Thermophoretic data, AN acquired the Fluorescence spectrophotometric data; Resources: PP, JJ, AG, and SW; Writing (Original Draft): MS, VT, and PP; Writing (Review & Editing): MS, AN, JJ, AG, SW, VT, and PP. Acknowledgments This work was supported by grants from the Estonian Research Council (Grant PRG 1289) to P.P. and from the Swedish Brain Foundation to A.G. and J.J. References Mahley RW, Apolipoprotein E (1988) Cholesterol Transport Protein with Expanding Role in Cell Biology. 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Nature 628(8006):154–161. https://doi.org/10.1038/s41586-024-07185-7 Parhizkar S, Holtzman DM (2022) APOE Mediated Neuroinflammation and Neurodegeneration in Alzheimer’s Disease. Semin Immunol 59:101594. https://doi.org/10.1016/j.smim.2022.101594 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8086961","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":543334865,"identity":"387d4f62-e11c-44b4-afcd-ab774f997486","order_by":0,"name":"Merlin Sardis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIie3QMWsCMRTA8RcO3hTrKgh3n6Dw5MBJva9iOLj5RodChUI6FVehpZ9BKUjHHAFdQme39pbuRSg9KNRwVSjSVMcO+S8JgR8vCYDP9z9javi9IQUjgDO7C46ZH8QQIJ5CdisBkyeQ8+ubF1U+QtK81fOiuv+MMLoqNjn0QhfpmhUpYUBMn7JcNxbUkYhpewpZ7CTrDJSQMATDSbMFMYm8G3DQYuwiz681SSJLiuqOEonNd0u+Lp1kjTVhM0tUY0zCTkFL9t/421vqi7XE3GCu+TJOJWZxm1PacU5ZLVlZyV4SmuDh7eMi7E8CXW74aBC5puxqHR7QEeDz+Xy+P9sCzPBTR3AjLigAAAAASUVORK5CYII=","orcid":"","institution":"Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia","correspondingAuthor":true,"prefix":"","firstName":"Merlin","middleName":"","lastName":"Sardis","suffix":""},{"id":543334866,"identity":"3ea0717a-858d-41b8-83a1-7ce7c4eb730d","order_by":1,"name":"Andra Noormägi","email":"","orcid":"","institution":"Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia","correspondingAuthor":false,"prefix":"","firstName":"Andra","middleName":"","lastName":"Noormägi","suffix":""},{"id":543334867,"identity":"f6b8317d-7510-4dc6-8d28-73044f5f27b2","order_by":2,"name":"Jüri Jarvet","email":"","orcid":"","institution":"Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden","correspondingAuthor":false,"prefix":"","firstName":"Jüri","middleName":"","lastName":"Jarvet","suffix":""},{"id":543334868,"identity":"ba087546-d8f6-4f44-8da1-71815a2aaecb","order_by":3,"name":"Astrid Gräslund","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYLCCBDYbxg0SxKtnBmlJI1ULA9thErTw958/9uFB2XnZ7dINzJ95GOrkCGqRuJHMPCPh3G3jnXMOsEnzMBw2JmzNDWZmhsS224kbbiSwMfMwHEhsIKRD/vxhkJZzIC1gh9UT1GJwIBmk5QBICwPQYcwJBN1leCPZmCHhXDLQLwfbJOcYHDYkaIvc+YOPGX+U2QFDrPnwhzcVdfIEbUECjEDzDUjRMApGwSgYBaMAJwAAdTI7XZSnx88AAAAASUVORK5CYII=","orcid":"","institution":"Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden","correspondingAuthor":true,"prefix":"","firstName":"Astrid","middleName":"","lastName":"Gräslund","suffix":""},{"id":543334869,"identity":"36f89b10-3639-4f2a-b226-f9222ccccda0","order_by":4,"name":"Sebastian K. 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16:31:56","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108517,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8086961/v1/0309aee3ea8d08778d457077.html"},{"id":95755110,"identity":"af2e15d1-917b-4a18-8e4a-90a84a62ab2b","added_by":"auto","created_at":"2025-11-12 16:31:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":239857,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8086961/v1/4c31ea2caf17b18a3e40b72b.png"},{"id":95801293,"identity":"e5ea12f3-037e-4695-af48-60a98d838175","added_by":"auto","created_at":"2025-11-13 08:24:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":350489,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8086961/v1/5f03b687a51eea4d67cfa432.png"},{"id":95805436,"identity":"49107e9a-3695-4210-be58-4db4c2c0e4b8","added_by":"auto","created_at":"2025-11-13 08:41:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1061534,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8086961/v1/3557c3c0-ea1f-4ad4-9365-da9387a67834.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eBinding of ApoE isoforms to Aβ peptides and effects on their fibrillization\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is the most prevalent neurodegenerative disorder, characterized by the occurrence of amyloid deposits in the brain composed of Aβ (amyloid-beta) peptides. The risk of developing late-onset Alzheimer\u0026rsquo;s disease (LOAD) is dependent on the isoforms of apolipoprotein E (ApoE) that individuals inherit. ApoE exists primarily in three isoforms: ApoE2 (Cys112, Cys158), ApoE3 (Cys112, Arg158), and ApoE4 (Arg112, Arg158) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. ApoE is co-localized with Aβ in senile plaques of AD and has been shown to bind tightly to immobilized Aβ peptide \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Notably, the presence of ApoE4 is recognized as the highest genetic risk factor for developing LOAD \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThere is substantial evidence that ApoE influences AD by affecting Aβ aggregation and clearance \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e; however, the molecular mechanism underlying this remains poorly understood. The direct interaction of ApoE with Aβ peptides has been studied mostly by ELISA; however, a few reports have used dual polarization interferometry (DPI) and surface plasmon resonance. According to ELISA, the dissociation constant values for ApoE-Aβ complexes are 48 nM (ApoE2), 63.7 nM (ApoE3), and 75.9 nM (ApoE4) (Yamauchi et al. 1999). Similar study using cell-derived ApoE (Sf9, HEK, and RAW cells) resulted in the following values: 13.3 nM (ApoE3), 13.9 nM (ApoE4) for Aβ1\u0026ndash;42 and 9.3 nM (ApoE3), 10.3 nM (ApoE4) for Aβ1\u0026ndash;40 (Tokuda et al. 2000). According to Sadowski et al., the dissociation constant of Aβ1\u0026ndash;40 complex with lipidated ApoE4 determined by ELISA is 14.7 nM (Sadowski et al. 2006). One study using DPI determined K\u003csub\u003ed\u003c/sub\u003e values for ApoE and Aβ1\u0026ndash;40 to be 251 nM (ApoE2), 40 nM (ApoE3), and 24.6 nM (ApoE4) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Surface plasmon resonance was used by Rahman et al. to determine K\u003csub\u003ed\u003c/sub\u003e values for ApoE and Aβ1\u0026ndash;42 protofibrils to be 3 nM\u003csup\u003e5\u003c/sup\u003e. Liu et al. used ELISA to map the binding regions between ApoE and Aβ, identifying residues 244\u0026ndash;272 on ApoE and 12\u0026ndash;28 on the Aβ peptide as the interacting sites \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Using a different approach, MD simulations, it was proposed that Aβ residues Asp1 and Asp23 might interact with cationic residues of ApoE, and thereby perturb its salt bridge network, especially in ApoE4 \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe effect of ApoE on fibrillization of Aβ has been widely studied; however, the results are conflicting. By some authors, it has been found that ApoE has a direct role in promoting and accelerating fibril formation of Aβ1\u0026ndash;40, where ApoE4 was more efficient than ApoE3 at enhancing amyloid formation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Sadowski et al. also showed that adding lipidated human ApoE4 significantly increased the amount of Aβ1\u0026ndash;40 fibrils \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Lipidated ApoE has been shown to increase Aβ oligomer levels in an isoform-dependent manner, with lipidated ApoE4 exerting the greatest effect on Aβ oligomer formation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Liao et al. claimed that ApoE-mediated plaque formation may be the result of ApoE aggregation, as evidenced by their observation that anti-human ApoE4 antibody binds nonlipidated and aggregated ApoE4 from amyloid plaques in mice, reducing Aβ deposition \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOn the contrary, Evans et al. showed that ApoE inhibits amyloid formation at substoichiometric levels \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, Garai et al. reported that at low concentrations, ApoE binds to and stabilizes Aβ oligomers, whereas at higher concentrations, it interacts with Aβ fibrils, stabilizing them and thereby inhibiting further fibril formation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In addition, ApoE has been shown to slow down the oligomerization of Aβ1\u0026ndash;40 in the solution \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The findings of Ghosh et al. also support the inhibitory effect, as they show that the Aβ1\u0026ndash;42 aggregation is delayed dramatically in the presence of stoichiometric concentrations of both lipid-free and lipidated ApoE forms, and that ApoE interacts rather with fibrillization intermediates than with the monomers of the Aβ peptide \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Recently, the same group reported that lipidated ApoE inhibits the elongation of Aβ1\u0026ndash;42 fibrils in an isoform-dependent manner, where ApoE2 and ApoE3 exhibited the strongest inhibitory effects, while secondary nucleation was largely unaffected \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. According to Islam et al., ApoE inhibits the process of fibril elongation and prevents amyloid maturation \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Xia et al. indicated that all ApoE isoforms associate with Aβ in the early stages of fibrillization and then fall away as fibrillization occurs \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It has also been shown that ApoE modulates the aggregation, clearance, and toxicity of Aβ in an isoform- and lipidation-specific way, by selectively removing non-lipidated ApoE4-Aβ co-aggregates and enhancing the clearance of toxic Aβ by glial cells \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Ghosh et al. found that both ApoE3 and ApoE4 also suppress at substoichiometric levels the aggregation of other proteins, for example, the AD-related proinflammatory protein S100A9, in a concentration-dependent manner \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMST is a powerful method for characterizing molecular interactions in solutions \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To date, MST has been used to study ApoE binding to complement regulator factor H \u003csup\u003e20\u003c/sup\u003e, various chemical probes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and α-synuclein \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In this study, we utilized MST to examine the interaction between Aβ peptides and ApoE and determined the effect of ApoE proteins on the fibrillization of Aβ1\u0026ndash;42 using the ThT assay. Our results indicate that Aβ peptides interact with ApoE at micromolar concentrations, whereas ApoE inhibits fibrillization of Aβ1\u0026ndash;42 at substoichiometric concentrations.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMicroscale thermophoresis (MST)\u003c/h2\u003e\u003cp\u003eThe binding of Aβ peptides to ApoE isoforms was studied using MST with fluorescently labelled ApoE (20 nM) isoforms (E2/E3/E4) as the target and Aβ1\u0026ndash;42 or Aβ1\u0026ndash;40 as the ligand.\u003c/p\u003e\u003cp\u003eThe obtained K\u003csub\u003ed\u003c/sub\u003e values remained in the range of 1\u0026ndash;2 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) except that for ApoE2 binding to Aβ1\u0026ndash;40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), characterized by the largest K\u003csub\u003ed\u003c/sub\u003e value, approx. 5 \u0026micro;M. The smallest K\u003csub\u003ed\u003c/sub\u003e value was below 1 \u0026micro;M and was observed for ApoE3 binding to Aβ1\u0026ndash;42.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e and RMSE values for binding of ApoE isoforms to Aβ1\u0026ndash;42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, a, b, c).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e# (Aβ1\u0026ndash;42)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eApoE2\u003c/p\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;RMSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eApoE3\u003c/p\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;RMSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eApoE4\u003c/p\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;RMSE\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e and RMSE values for binding of ApoE isoforms to Aβ1\u0026ndash;40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, d, e, f).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e# (Aβ1\u0026ndash;40)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eApoE2\u003c/p\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;RMSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eApoE3\u003c/p\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;RMSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eApoE4\u003c/p\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;RMSE\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOur results show that the K\u003csub\u003ed\u003c/sub\u003e values for ApoE-Aβ complexes determined by MST experiments show significantly weaker affinity than those estimated earlier by ELISA and DPI. The observed difference can be attributed to the nature of the measurements: MST measurements determine interactions in solutions, whereas ELISA and DPI evaluate the affinity of ApoE to the peptide immobilized on a solid surface. It is well known that affinity values vary depending on the experimental conditions and the technique used for the measurements \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The current MST results indicate that the interaction of ApoE with Aβ peptide occurs at supraphysiological micromolar concentrations, suggesting that ApoE interaction with Aβ peptides is unlikely to occur under physiological conditions. Measured K\u003csub\u003ed\u003c/sub\u003e values varied only slightly, with differences ranging from three to fourfold, depending on the type of ApoE or Aβ isoform. ApoE4 exhibits the weakest binding to Aβ1\u0026ndash;42 but the strongest binding to Aβ1\u0026ndash;40. The experiments were conducted using lipid-free ApoE, which can bind nonpolar substrates, and it could be suggested that in the case of lipidated ApoE, the K\u003csub\u003ed\u003c/sub\u003e values may be even higher. Our findings align with those of Verghese et al., who used multiple biochemical and analytical techniques to demonstrated minimal binding of lipidated ApoE to Aβ peptide in physiological fluids, with a slight increase in binding observed when the lipidation level was reduced \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFluorescence spectrophotometry\u003c/h3\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1762964882.png\" style=\"width: 731px;\"\u003e\u003c/p\u003e\u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e values for ApoE, determined from the inhibition of Aβ fibrillization, were lower than the K\u003csub\u003ed\u003c/sub\u003e values. Notably, the inhibition occurred already at low substoichiometric concentrations: 50 times less ApoE reduces the rate of Aβ1\u0026ndash;42 fibrillization by 1.5 to 2.5 times. The inhibitory effect of ApoE on the fibrillization rate of Aβ1\u0026ndash;42 may be explained by ApoE blocking of Aβ1\u0026ndash;42 fibrillization sites. Recently, Dasadhikari et al. reported that the lipidated ApoE inhibits Aβ1\u0026ndash;42 fibril elongation, while secondary nucleation remains largely unaffected \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Single fibril studies confirmed that the inhibition of the elongation rate is proportional to the binding of ApoE to the terminal ends of the fibrils. The affinity constants of ApoE isoforms for fibril ends were isoform-specific, with ApoE4 exhibiting fourfold weaker binding compared to ApoE2 and ApoE3 \u003csup\u003e16\u003c/sup\u003e. In our study, we observed rather similar inhibitory effects of different ApoE isoforms on Aβ1\u0026ndash;42 fibril elongation rate, whereas ApoE4 showed only slightly stronger inhibition. Fibrillization experiments, similarly to MST experiments, were performed with lipid-free ApoE, and therefore, the results may be different in the case of lipidated ApoE forms.\u003c/p\u003e\u003cp\u003eCurrent results show that ApoE has a weak ability to interact with Aβ peptides; however, its ability to interact with intermediates of Aβ fibrillization is substantially higher. Therefore, ApoE can block the fibrillization of Aβ peptides, where the effects of different ApoE isoforms are quite similar. According to our results, the isoform-specific behavior of ApoE in the pathogenesis of AD is not linked to its interaction with Aβ monomers or aggregation intermediates. Still, it might be connected to its other interactions.\u003c/p\u003e\u003cp\u003eThe ApoE genotype may contribute to AD pathogenesis through several other distinct mechanisms. One hypothesis is related to LDL receptor-related protein 1 (LRP1), which is involved in the internalization and degradation of Aβ \u003csup\u003e25 26\u003c/sup\u003e. ApoE has been shown to disrupt Aβ clearance from the brain \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and blocking the ApoE/Aβ interactions resulted in enhanced Aβ clearance from the brain and decreased plaque deposition \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, ApoE isoforms may influence Aβ metabolism by competing for the same clearance pathways in the brain \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Alternatively, there might occur ApoE isoform-specific microglial lipid droplet accumulation, which leads to Tau phosphorylation and neurotoxicity in an ApoE-dependent manner \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It is also known that ApoE4 increases tau pathogenesis and leads to increased astroglial and microglial-mediated persistent inflammation, which leads to significant neurodegeneration in the presence of ApoE4 \u003csup\u003e30\u003c/sup\u003e. The mystery of how slight modifications in the protein sequence of ApoE isoforms affect the propensity of AD remains to be established.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eLyophilized Aβ1\u0026ndash;40 and Aβ1\u0026ndash;42 were purchased from r-Peptide (Watkinsville, GA, USA); ApoE isoforms (E2/E3/E4) were purchased from AlexoTech AB (Ume\u0026aring;, Sweden). The integrity of the proteins were verified by MALDI-MS.\u003c/p\u003e\u003cp\u003e1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), HEPES, NaCl, NaOH, glycerol, NH\u003csub\u003e4\u003c/sub\u003eOH, and Thioflavin-T (ThT) were purchased from Sigma-Aldrich.\u003c/p\u003e\u003cp\u003ePluronic F-127, labelling buffer (LB-NHS), RED-NHS 2nd Generation dye, B-column, and DMSO were included in Protein Labelling Kit RED-NHS 2nd Generation (MO-L011) and purchased from Nanotemper (Nano Temper Technologies, M\u0026uuml;nchen, Germany).\u003c/p\u003e\n\u003ch3\u003eSample preparation\u003c/h3\u003e\n\u003cp\u003eLyophilized Aβ1\u0026ndash;40 and Aβ1\u0026ndash;42 were solubilized in HFIP at a concentration of 100 \u0026micro;M, divided into aliquots, and the HFIP was evaporated under vacuum. The tubes with Aβ film were stored at -80\u0026deg;C. HFIP treatment is used to disassemble peptide aggregates into monomers.\u003c/p\u003e\u003cp\u003eApoE isoforms (E2/E3/E4) were dissolved in a 5 mM NaOH solution to a final concentration of 50 \u0026micro;M, divided into aliquots, and stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eMicroscale thermophoresis (MST)\u003c/h3\u003e\n\u003cp\u003eApoE isoforms were diluted to a final concentration of 10 \u0026micro;M with labelling buffer (LB-NHS in MO-L011, Nanotemper), and labelled with NHS reactive dye (30 \u0026micro;M, dissolved in DMSO) from Protein Labelling Kit RED-NHS 2nd Generation (MO-L011; Nanotemper, M\u0026uuml;nchen, Germany). The labelling reaction was carried out for 30 min at 25\u0026deg;C in the dark, the mixture was applied to the column according to the manufacturer\u0026rsquo;s instructions, and eluted with 50 mM HEPES, 150 mM NaCl, pH\u0026thinsp;=\u0026thinsp;7.4, 10% glycerol, 0.1% Pluronic F-127. 10% glycerol was added to the buffer to decrease the aggregation of ApoE. Labelling efficiency was estimated using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) to be in the range of DOL\u0026thinsp;=\u0026thinsp;0,5\u0026thinsp;\u0026minus;\u0026thinsp;1. Labelled ApoE isoforms were divided into aliquots and stored at -20\u0026deg;C. Before use, the labelled ApoE isoforms were thawed, diluted to 40 nM in 100 mM HEPES, 300 mM NaCl, pH 7.4, 20% glycerol, 0.2% Pluronic F-127, and centrifuged at 21,000 g, 4\u0026deg;C, for 10 min.\u003c/p\u003e\u003cp\u003eHFIP-treated Aβ1\u0026ndash;42 and Aβ1\u0026ndash;40 aliquots were dissolved in 5 mM NaOH to a final concentration of 40 \u0026micro;M and 100 \u0026micro;M, respectively, incubated for 10 min on ice, and centrifuged for 10 min, 4\u0026deg;C, 21,000 g before use. Since amyloid β tends to aggregate at neutral pH, the 16-point serial dilutions of Aβ1\u0026ndash;40 (100 \u0026micro;M) and Aβ1\u0026ndash;42 (40 \u0026micro;M) were made in 5 mM NaOH. Samples for measurement were prepared by mixing 10 \u0026micro;l of a 16-point serial dilution of Aβ1\u0026ndash;40 (100 \u0026micro;M) or Aβ1\u0026ndash;42 (40 \u0026micro;M) with an equal amount of labelled ApoE isoforms (40 nM) with 10 \u0026micro;l of Aβ1\u0026ndash;40 or Aβ1\u0026ndash;42 to the final concentration of Aβ1\u0026ndash;40 and Aβ1\u0026ndash;42 ranging from 50 \u0026micro;M to 3 nM and 20 \u0026micro;M to 1,3 nM, respectively. Samples were measured in 50 mM HEPES, 150 mM NaCl, pH\u0026thinsp;=\u0026thinsp;7.4, containing 10% glycerol and 0.1% Pluronic F-127. After mixing, the samples were instantly loaded into Standard capillaries, inserted into the Monolith NT 115 system (Nano Temper Technologies, M\u0026uuml;nchen, Germany), and measured using 20% LED and low MST intensity (20% IR-laser power), with temperature control at 23\u0026deg;C. Results were analyzed with MO Affinity Analysis software and fitted by the K\u003csub\u003ed\u003c/sub\u003e equation provided by the software. MST-on time used for analysis was 20 seconds. The K\u003csub\u003ed\u003c/sub\u003e values, together with root mean square error (RMSE), were estimated using MO Affinity Analysis Software.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFluorescence spectrophotometry\u003c/h2\u003e\u003cp\u003eHFIP-treated Aβ1\u0026ndash;42 was dissolved in 0.02% NH\u003csub\u003e4\u003c/sub\u003eOH, incubated 10 min on ice, and diluted with 40 mM HEPES, 200 mM NaCl, pH\u0026thinsp;=\u0026thinsp;7.4 to a final concentration of 5 \u0026micro;M Aβ1\u0026ndash;42, 20 mM HEPES, 100 mM NaCl, pH\u0026thinsp;=\u0026thinsp;7.4.\u003c/p\u003e\u003cp\u003eFibrillization of Aβ1\u0026ndash;42 was studied using fluorescent ligand ThT, whose fluorescence intensity at 480 nm (excitation at 440 nm) is increased upon binding to amyloid fibrils. ThT fluorescence was monitored on a Perkin Elmer (Waltham, MA, USA) LS55 fluorescence spectrophotometer in a 500 \u0026micro;L cuvette by constant stirring at 40\u0026deg;C in the presence of 5 \u0026micro;M ThT. To study the effect of ApoE isoforms on Aβ1\u0026ndash;42 fibrillization, Aβ1\u0026ndash;42 fibrillization curves in the absence and presence of different concentrations of ApoE2, ApoE3, and ApoE4 were determined and fitted to the Boltzmann Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) by the program Origin 8.5 (OriginLab Corporation, USA):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{y}=\\frac{{\\text{A}}_{1}-{\\text{A}}_{2}}{1+{e}^{\\left(t-{t}_{0}\\right)\\bullet\\:k}}\\:+{\\text{A}}_{2}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere A1 is the initial fluorescence intensity level, A2 corresponds to the fluorescence at maximal fibrillization level, t0 is the time t when fluorescence is reached half maximum, and k is the apparent rate constant for the growth of fibrils.\u003c/p\u003e\u003cp\u003eThe half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values were calculated from the effects of ApoE isoforms on the apparent rate constant of fibril formation (IC\u003csub\u003e50 k\u003c/sub\u003e) according to hyperbolic dose-response curves:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{y}={\\text{A}}_{2}-\\frac{{A}_{2}\\text{*}\\text{c}}{{\\text{I}\\text{C}}_{50}+\\text{c}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere y is the fluorescence intensity of ThT, A2 is the maximum value of ThT fluorescence, c is the concentration of the test substance, and IC\u003csub\u003e50\u003c/sub\u003e is the concentration that reduces ThT fluorescence by 50%. Normalization of data and nonlinear regression analysis were carried out using the program Origin 8.5.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eConceptualization: VT and PP; Investigation: MS acquired the Microscale Thermophoretic data, AN acquired the Fluorescence spectrophotometric data; Resources: PP, JJ, AG, and SW; Writing (Original Draft): MS, VT, and PP; Writing (Review \u0026amp; Editing): MS, AN, JJ, AG, SW, VT, and PP.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the Estonian Research Council (Grant PRG 1289) to P.P. and from the Swedish Brain Foundation to A.G. and J.J.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMahley RW, Apolipoprotein E (1988) Cholesterol Transport Protein with Expanding Role in Cell Biology. 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Semin Immunol 59:101594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.smim.2022.101594\u003c/span\u003e\u003cspan address=\"10.1016/j.smim.2022.101594\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Estonian Research Council","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, amyloid β peptide, apolipoprotein E, microscale thermophoresis, fibrillization, ThT assay","lastPublishedDoi":"10.21203/rs.3.rs-8086961/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8086961/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is the most widespread neurodegenerative disease, strongly linked to amyloid depositions in the brain consisting of amyloid beta (Aβ) peptides. The likelihood of developing late-onset Alzheimer\u0026rsquo;s disease (LOAD) is influenced by the specific isoforms of apolipoprotein E (ApoE), with ApoE4 being the strongest known genetic risk factor for LOAD. Strong evidence suggests that ApoE impacts AD by modulating Aβ aggregation and clearance, although the precise molecular mechanisms remain incompletely understood. Microscale thermophoresis (MST) is a powerful technique for characterizing molecular interactions in solution, which has been used to determine various binding constants, although not the binding of ApoE to Aβ peptides. MST results show that ApoE isoforms bind Aβ1\u0026ndash;40 and Aβ1\u0026ndash;42 with low micromolar affinity. For Aβ1\u0026ndash;42, ApoE3 shows the strongest binding (K\u003csub\u003ed\u003c/sub\u003e = 0.72 \u0026micro;M) and ApoE4 the weakest (K\u003csub\u003ed\u003c/sub\u003e = 2.80 \u0026micro;M). For Aβ1\u0026ndash;40, ApoE4 shows the strongest binding (K\u003csub\u003ed\u003c/sub\u003e = 1.59 \u0026micro;M) and ApoE2 the weakest (K\u003csub\u003ed\u003c/sub\u003e = 5.29 \u0026micro;M). The MST results show that ApoE interacts with Aβ peptides at supraphysiological peptide concentrations. However, ApoE inhibited the fibrillization of Aβ1\u0026ndash;40 peptide at sub-stoichiometric concentrations, which might be related to blocking Aβ fibril elongation in vivo. The estimated IC\u003csub\u003e50\u003c/sub\u003e values indicate that ApoE4 has slightly stronger and ApoE2 has the lowest inhibitory effect on Aβ1\u0026ndash;42 fibrillization.\u003c/p\u003e","manuscriptTitle":"Binding of ApoE isoforms to Aβ peptides and effects on their fibrillization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 16:31:51","doi":"10.21203/rs.3.rs-8086961/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12215e6f-0b6a-4e92-9d85-77cc2dd3b8e7","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57802612,"name":"Biophysics"}],"tags":[],"updatedAt":"2025-11-12T16:31:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 16:31:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8086961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8086961","identity":"rs-8086961","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}