Interactions between BIM Protein and Beta-Amyloid May Reveal a

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Interactions between BIM Protein and Beta-Amyloid May Reveal a Crucial Missing Link between Alzheimer’s Disease and Neuronal Cell Death Ravit Malishev,† Sukhendu Nandi,‡ Dariusz Śmiłowicz,‡ Shamchal Bakavayev,§ Stanislav Engel,§,∥ Nir Bujanover,∥ Roi Gazit,∥ Nils Metzler-Nolte,‡ and Raz Jelinek*,†

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Department of Chemistry and Ilse Katz Institute for Nanotechnology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ‡ Inorganic Chemistry I − Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany § Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel ∥ National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel S Supporting Information *

ABSTRACT: Extensive neuronal cell death is among the pathological hallmarks of Alzheimer’s disease. While neuron death is coincident with formation of plaques comprising the beta-amyloid (Aβ) peptide, a direct causative link between Aβ (or other Alzheimer’s-associated proteins) and cell toxicity is yet to be found. Here we show that BIM-BH3, the primary proapoptotic domain of BIM, a key protein in varied apoptotic cascades of which elevated levels have been found in brain cells of patients afflicted with Alzheimer’s disease, interacts with the 42-residue amyloid isoform Aβ42. Remarkably, BIM-BH3 modulated the structure, fibrillation pathway, aggregate morphology, and membrane interactions of Aβ42. In particular, BIM-BH3 inhibited Aβ42 fibril-formation, while it simultaneously enhanced protofibril assembly. Furthermore, we discovered that BIM-BH3/Aβ42 interactions induced cell death in a human neuroblastoma cell model. Overall, our data provide a crucial mechanistic link accounting for neuronal cell death in Alzheimer’s disease patients and the participation of both BIM and Aβ42 in the neurotoxicity process. KEYWORDS: Beta-amyloid, BIM, Bcl-3 homology 3, mitochondria, apoptosis, amyloid/membrane interaction

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INTRODUCTION Alzheimer’s disease (AD) is a devastating and incurable disease, more prevalent in light of increasing human longevity, and which imposes considerable emotional and economical toll. Intense research over the past decades has not resolved yet the exact molecular-mechanistic aspects of AD. Phenomenologically, AD is characterized by extensive neuronal cell death in the brain that correlates with deposition of abundant fibrillar plaques, primarily comprising the beta-amyloid (Aβ) peptide.1,2 Aβ is a product of sequential cleavage of amyloid βprotein precursor (APP) by the proteolytic activities of βsecretase and γ-secretase enzymes. It is yet unknown whether the fibrillar Aβ deposits actually partake in toxicity mechanisms, or whether the plaques constitute a side product and they do not inflict neuronal damage. Indeed, numerous studies have reported that Aβ oligomers and prefibrillar species are the actual cytotoxic agents,3,4 and that the primary adverse physiological events induced by the oligomeric species occur at the cell membrane.5,6 Recent evidence, for example, suggests © XXXX American Chemical Society

that extracellular Aβ oligomers interact with cell surfaces, leading to functional disruption of various receptors resulting in the abnormal activation of signaling pathways.4,7 Notably, many studies have indicated that Aβ also accumulates intracellularly;8,9 however the physiological significance of these observations is yet to be resolved. Recent studies have reported that intracellular Aβ is specifically localized within the mitochondria.10−12 Aβ itself can be imported into mitochondria via the translocase within the outer mitochondrial membranes and is consequently found within the mitochondrial cristae in the inner mitochondrial membrane. Mitochondria are the primary cellular hosts of apoptotic proteins and as such constitute the organelle most responsible for initiating apoptotic cascades−the critical programmed cell death processes.13,14 Intriguingly, both Received: March 25, 2019 Accepted: May 29, 2019 Published: May 29, 2019 A

DOI: 10.1021/acschemneuro.9b00177 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. Binding of the proapoptotic BH3 domain of the BIM protein to Aβ42. SPR assay employing immobilized Aβ42 monomers and different concentrations of the BIM-BH3 peptide. (A) SPR sonograms recorded for different BIM-BH3 concentrations. (B) Corresponding dose−response curve. Kd = 7.1 × 10−6 M.

Figure 2. Fibrillation kinetics of Aβ42 with and without the BIM-BH3 peptide. (A) ThT fluorescence curves recorded in the absence or presence of different BIM-BH3 concentrations: 10 μM Aβ42 alone (i); 10 μM Aβ42 + 5 μM BIM-BH3 (ii); 10 μM Aβ42 + 10 μM BIM-BH3 (iii); 10 μM Aβ42 + 20 μM BIM-BH3 (iv). (B) TEM images of Aβ42 samples after 14 h incubation in the absence (i) or presence (ii−iv) of BIM-BH3.

BIM-BH3 and Aβ42 dramatically increased neuronal cell toxicity, likely due to enhanced membrane interactions of BIM-BH3-induced Aβ42 prefibrillar species. Overall, our data might reveal a mechanistic link between BIM, intracellular Aβ42 accumulation, and neuronal cell death.

monomeric and oligomeric Aβ have been identified in mitochondria of neurons affected by AD.15−17 Moreover, Aβ was shown to interact with mitochondrial proteins;18 such interactions have been implicated in increased free radical production,19 lower cytochrome c oxidase activity,20 inhibition of mitochondrial ATP production,21 and overall damage to mitochondrial structures.22 Indeed, experimental evidence suggests that structural and functional abnormalities in mitochondria constitute intrinsic facets of AD.15 While the relationship between mitochondrial dysfunctions and Aβ is still unclear, several reports have indicated elevated levels of apoptotic proteins residing primarily in mitochondria in Alzheimer’s patients.23−27 Specifically, high concentrations of B-cell lymphoma (BCL-2) apoptotic proteins, including BIM, BAK, and BAD, have been reported within afflicted neuronal cells.23−27 Interestingly, other studies have reported high abundance of BIM, a prominent intracellular protein participating in varied apoptotic cascades, in cells associated with other amyloidogenic pathologies including type-II diabetes28,29 and Huntington’s disease.30−32 These observations might point to distinct relationships between Aβ and apoptosis. Indeed, association between the 1−42 isoform of Aβ (Aβ42) and BIM has been reported.18,33,34 Whether these observations have physiological or clinical significance has not been determined. Here, we show that the Bcl-2 homology 3 (BH3) domain, the primary pro-apoptotic domain of BIM, significantly modulates structural transformations and fibrillation properties of Aβ42. Importantly, we found that interactions between

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RESULTS Binding of Aβ42 to BIM-BH3 and Structural Transformations of the Amyloid Peptide. Figure 1 depicts surface plasmon resonance (SPR) measurements, in which Aβ42 monomers were immobilized on the sensor chip and the BIM-BH3 segment was added in different concentrations. The SPR sensograms (Figure 1A) and corresponding dose− response curve (Figure 1B) indicate binding between Aβ42 and BIM-BH3, with a calculated Kd of 7.1 × 10−6 M. This binding constant is similar in magnitude to values previously reported for immobilized Aβ42 interacting with peptides and small molecules.35,36 While Figure 1 reveals notable interactions between the proapoptotic BIM-BH3 peptide and Aβ42, we further examined the effect of BIM-BH3 upon Aβ42 aggregation using thioflavin-T (ThT) fluorescence (Figure 2A). ThT is a widely used dye employed for illuminating protein aggregation.37,38 Specifically, ThT fluorescence increases upon association of the dye within β-strand aggregates and fibrillar species.37 The ThT fluorescence data in Figure 2 attest to a dramatic effect of BIM-BH3 upon Aβ42 aggregation. The ThT fluorescence curve of Aβ42 alone (at a concentration of 10 μM) shows very small fluorescence increase during the B


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Figure 3. Structural transformations of Aβ42 in the presence of BIM-BH3 peptide. (A) Time-dependent secondary structures of Aβ42 monitored by CD spectroscopy in the absence or presence different BIM-BH3 concentrations: 25 μM Aβ42 alone (black spectra); 25 μM Aβ42 + 12.5 μM BIM-BH3 (red spectra); 25 μM Aβ42 + 25 μM BIM-BH3 (blue spectra). (B) Visualization and kinetic analysis of peptide aggregation by AFM: 25 μM Aβ42 alone (left column); 25 μM Aβ42 + 12.5 μM BIM-BH3 (middle column); 25 μM Aβ42 + 25 μM BIM-BH3 (right column). Incubation times of the peptides are indicated.

further reveals that smaller fibrillar aggregates form in solutions containing higher ratios between BIM-BH3 and Aβ42 (Figure 2B,iii,iv). The TEM results in Figure 2B complement the ThT fluorescence data (Figure 2A), as the abundant smaller Aβ42 aggregates forming upon BIM-BH3 addition provide pronounced surface area for ThT association and concomitant increased fluorescence of the dye.37 To decipher the effects of BIM-BH3 on the structural transformations of Aβ42 in aqueous solution and the kinetics of Aβ42 aggregation, we carried out circular dichroism (CD) and atomic force microscopy (AFM) experiments (Figure 3). Figure 3A presents CD spectra of Aβ42 recorded at different times after dissolving the peptide in water, with and without coincubation with the BIM-BH3 peptide. The time-dependent CD signatures of Aβ42 alone (Figure 3A, black spectra) reveal gradual spectra shift and appearance of the negative peak at around 215 nm and positive shoulder at 195 nm, corresponding to the random coil-to-β sheet transformation of the peptide.42 Similar CD traces were recorded when a low concentration of BIM-BH3 was coincubated with Aβ42 (BH3:Aβ42 mol ratio of 1:0.5; Figure 3A, red spectra). However, significant acceleration of β-sheet assembly of Aβ42 was clearly apparent in a sample comprising 1:1 mol ratio between Aβ42 and BIM-BH3 (Figure 3A, blue spectra). Importantly, the CD spectra in Figure 3A constitute the net secondary structures of Aβ42, after subtraction of the corresponding spectra of BIM-BH3 alone (the CD traces of

measurement window (Figure 2A, black curve). This behavior may be explained by the fact that relatively small differences in Aβ42 concentrations could result in changes of the aggregation rates; this phenomenon was shown previously in the case of the amyloidogenic peptide islet amyloid polypeptide protein (IAPP).39,40 Significantly enhanced ThT fluorescence evolution, however, was recorded upon coincubation of the dye with BIMBH3/Aβ42 mixtures. Notably, a direct relationship was apparent in Figure 2A between the degree of ThT fluorescence intensity and BIM-BH3 concentration. Since BIM-BH3 alone did not give rise to ThT fluorescence (data not shown), the dramatic increase in ThT fluorescence reflects the occurrence of distinct Aβ42 aggregation pathways and/or enhanced fibrillation of the peptide, that were specifically induced by BIM-BH3. The transmission electron microscopy (TEM) analysis in Figure 2B further illuminates the dramatic effects of Aβ42/ BIM-BH3 interactions upon the morphology and abundance of peptide aggregates formed. The TEM images in Figure 2B were recorded after 14 h incubation for the respective Aβ42/ BIM-BH3 mixtures analyzed in the ThT experiments (i.e., Figure 2A). Aβ42 alone produces the widely observed elongated fibrils,41 dispersed in relatively low abundance upon the TEM grid (Figure 2B,i). Ubiquitous thicker and shorter fibrillar species appear, however, following coincubation of Aβ42 with BIM-BH3 (Figure 2B,ii). The TEM analysis C


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Figure 4. Interactions of BIM-BH3/Aβ42 mixtures with membrane vesicles. Effects of the peptides on bilayer dynamics probed using fluorescence anisotropy measurements. Shown are the fluorescence anisotropy values of DPH embedded in (A) DOPC/Chol/Sph vesicles (0.67:0.25:0.08 mol ratio) or (B) DOPC/cardiolipin vesicles (0.96:0.04 mol ratio). Fluorescence anisotropy was measured following addition of 25 μM Aβ42 alone or in the presence of BIM-BH3: (I) 25 μM Aβ42 + 12.5 μM BIM-BH3; (II) 25 μM Aβ42 + 25 μM BIM-BH3; (III) BIM-BH3 12.5 μM; (IV) BIMBH3 25 μM. Results are presented as means ± standard error of the mean (SEM) using seven replicates. *p < 0.001 compared to control vesicles of the particular experiment.

Particularly striking is the observation that the Aβ42 protofibrils were still the predominant species in the BIMBH3/Aβ42 mixtures after 48 h, while mature fibrils could be hardly detected (Figure 3B, bottom row, middle and right images). This result stands in contrast to the AFM results of bare Aβ42, which featured almost exclusively long, mature fibrils (e.g., bottom left panel, Figure 3B). Overall, the microscopy analyses in Figures 2B and 3B complement the CD and ThT data (Figures 2A and 3A, respectively), underscoring the pronounced effects of the proapoptotic BIM-BH3 peptide upon the structural transformations, aggregation pathway, and fibril morphologies of Aβ42. Numerous studies have proposed that distinct prefibrillar species assembled during the aggregation process of Aβ42 are intimately linked to the biological effects and toxicity profile of the peptide, and to the pathology of Alzheimer’s disease in general.4,45,46 In particular, Aβ42 oligomers and small prefibril aggregates are believed to constitute major toxicity determinants, possibly through membrane interactions and concomitant disruption of the lipid bilayer.47−49 Accordingly, we examined whether, and to what degree, the proapoptotic BIMBH3 peptide affects the functional and biological properties of Aβ42 (Figures 4 and 5). Importantly, the A11 antibody assay50 confirmed the presence of abundant oligomeric Aβ42 species in the BH3/Aβ42 mixtures (Figure 2,SI). Effect of BIM-BH3 upon Membrane Interactions of Aβ42. To evaluate membrane interactions of Aβ42 and the BIM-BH3/Aβ42 mixtures (at different mole ratios between the two peptides) we carried out fluorescence anisotropy experiments, presented in Figure 4, utilizing lipid vesicles, which also contained the fluorescent dye diphenylhexatriene (DPH). The fluorescence anisotropy of bilayer-embedded DPH has been employed as a sensitive measure of lipid fluidity and the effect of membrane-active molecules upon lipid mobility in the bilayer.51,52 The fluorescence anisotropy data in Figure 4 were recorded using vesicles comprising either dioleylphosphatidylcholine (DOPC), cholesterol (chol), and sphingomyelin (Sph), designed to mimic the plasma membrane, or vesicles prepared from DOPC and cardiolipin (CL), mimicking the mitochondrial membrane which is the main cellular compartment hosting BIM-BH3.53,54 Importantly, AFM experiments shown in Figure 3,SI confirmed that similar Aβ42 fibrillation

BIM-BH3 alone indicate a mostly random coil and a certain percentage of helical conformation (Figure 1,SI)).43 All the signals resulting from the BIM-BH3 were subtracted from the corresponding spectra. The dramatic BH3-induced remodeling of Aβ42 structure occurred, in fact, immediately after codissolution of the two peptides, apparent in the spectral shift of the blue spectrum at the nominal time t = 0 (Figure 3A). The CD data, attesting to BH3-promoted β-sheet formation of Aβ42 (Figure 3A), are consistent with the enhanced ThT fluorescence and TEM data in Figure 2, which demonstrated rapid formation of Aβ42 aggregates upon coincubation with BIM-BH3. To further probe the impact of the proapoptotic BIM-BH3 segment upon the fibrillation process of Aβ42, we carried out microscopy experiments designed to visualize the aggregate morphologies and their time evolution (Figure 3B). Corroborating the ThT and CD experiments (Figure 2A and 3A), the atomic force microscopy (AFM) images in Figure 3B demonstrate that BIM-BH3 significantly modulated Aβ42 fibril assembly. Aβ42 alone formed small prefibrillar aggregates short time after dissolution (Figure 3B, left column, top image). The small aggregates (oligomers and protofibrils44) likely constitute nucleation sites for further fibril growth, yielding mature fibrils after longer incubation times (Figure 3B, left column, middle and bottom panels). The AFM images in Figure 3B (middle and right columns) reveal that coincubation of BIM-BH3 with Aβ42 gave rise to abundant prefibrillar aggregates. Moreover, the pro-apoptotic BIM-BH3 domains affected Aβ42 aggregate morphologies and the extent of elongated fibril growth (Figure 3B). Specifically, ubiquitous small aggregates are apparent in the BIM-BH3/ Aβ42 mixtures, at both mole ratios, after a short, 10 min incubation (Figure 3B, top middle and right images). This result is consistent with the ThT fluorescence data in Figure 2A, pointing to rapid Aβ42 aggregation induced by the BIMBH3 segment. Abundant, short protofibril structures are apparent in the BIM-BH3/Aβ42 mixtures incubated for 24 h, and particularly in the mixed-peptide samples incubated for 48 h (Figure 3B, middle and right images, middle and bottom rows). These protofibril configurations appear larger and morphologically different than the prefibrillar species of Aβ42 alone (Figure 3B, left column). D


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Figure 5. BIM-BH3 and Aβ42 have cooperative proapoptotic function. (A) Apoptotic activity of Aβ42 in the absence or presence of BIM-BH3 was studied by annexin V and PI staining. SH-SY5Y cells were incubated at 37 °C in cell medium for 24 h. The controls were treated by medium with or without BIM-BH3, as indicated, for additional 48 h. After treatments, cells were harvested and stained using Annexin V−FITC and PI. FACS analysis is demonstrated in the representative plots (left panels) and quantified in the histograms for live cells (blue bars), apoptotic cells (red bars), and necrotic/dead (green bars); Data were collected from triplicates, showing averages ± SD. (B) Morphological changes in SH-SY5Y cells incubated with 15 μM Aβ42 and the indicated Aβ42 + BIM-BH3 mixtures at 15 and 45 μM, respectively.

vesicles, reflecting the presumed affinity of the peptide to cholesterol-rich lipid raft domains.56,57 Previous studies have shown that cholesterol-enriched lipid rafts play important roles in accelerating plaque formation and membrane disruption.58 Moreover, one has to consider the critical micellar concentration (cmc) of phospholipid components of the vesicles produced. Specifically, previous analyses have correlated the lipids’ cmc and peptide fibrils induced by free lipids in

pathways occurred in the presence of both types of vesicles as in buffer (i.e., Figure 3). Figure 4 reveals that, in both vesicle models, the proapoptotic BIM-BH3 peptide enhanced bilayer interactions of Aβ42. Upon addition of Aβ42 alone, the fluorescence anisotropy of DPH increased due to insertion of Aβ42 into the bilayer thereby restricting the mobility of the phospholipid acyl chains.55 The higher Aβ42-induced anisotropy was particularly significant in the DOPC/Chol/Sph E


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Figure 6. Proposed model for the physiological implications of BIM-BH3/Aβ42 interactions. Top: Aβ42 monomers cleaved by APP (red cylinder) assemble into membrane-active oligomeric and prefibril species, which subsequently assemble into mature fibrils. Bottom: BIM-BH3 binds to Aβ42, promoting its oligomerization and prefibrils responsible for membrane disruption and cytotoxicity.

equilibrium with the assembled vesicles.59,60 In this context, the different extents of fibrils formed in the two lipid mixtures might affect the degree of Aβ42-induced fluidity of the bilayers. A particularly notable result in Figure 4 is the pronounced DPH fluorescence anisotropy induced upon adding BIM-BH3/ Aβ42 mixtures to the vesicles, in comparison to Aβ42 alone. BIM-induced higher anisotropy was apparent in the PC/chol/ Sph vesicle model (Figure 4A, bars (I) and (II)) and particularly in case of the mitochondrial membrane model (Figure 4B, bars (I) and (II)). The enhanced anisotropy values indicate that BIM-BH3 promoted greater membrane internalization of Aβ42. This result is consistent with the apparent BH3-induced Aβ42 protofibril formation (e.g., Figure 3), as Aβ42 oligomers and prefibrillar aggregates have been shown to exhibit significant membrane interactions.61−63 It should be noted that BIM-BH3 alone exhibited negligible effects upon bilayer fluidity (Figure 4, bars iii and (iv), confirming that the enhanced bilayer interactions of Aβ42 were due to the impact of the coadded BIM-BH3 peptide. Impact of BIM-BH3 upon Aβ42-Induced Neuronal Cell Death. While Figure 4 shows that the distinct effects of the proapoptotic BIM-BH3 domain upon Aβ42 structural features and fibril formation likely induced greater membrane interactions and bilayer internalization of Aβ42, a crucial question concerns the biological significance of BIM-BH3/ Aβ42 interactions. Indeed, Figure 5 attests to the dramatic consequences of coincubating BIM-BH3 and Aβ42 upon neuronal cell viability. Figure 5A depicts an PI-annexin flow cytometry apoptosis assessment utilizing the SH-SY5Y neuroblastoma cell line.64,65 In the absence of BIM-BH3, 15 μM Aβ42 exhibited a toxic effect, reducing cell viability to around 60%, while in parallel affecting an increase in the populations of apoptotic and necrotic cells (Figure 5A, top row). Furthermore, coincubation of Aβ42 with increasing concentrations of BIM-BH3 (Aβ42/BIM-BH3 at 1:1, 1:2, and 1:3 mol ratios, respectively) induced higher toxicity, decreasing cell viability to around 40% at the highest BIM-BH3 concentration, and significantly increasing the fractions of apoptotic and

necrotic cells (Figure 5A, three bottom rows).66 Importantly, the BIM-BH3 peptide alone did not produce toxic effects. The optical microscopy images in Figure 5B similarly highlight the enhanced toxic effects induced upon coincubation of BIM-BH3 and Aβ42 with the SH-SY5Y neuroblastoma cells. While addition of Aβ42 (15 μM) to the cells induced apoptotic cell death, reflected in the cell shape transformations to more spherical appearance (Figure 5B, middle image), coaddition of Aβ42 together with the proapoptotic BIM-BH3 peptide gave rise to considerably more extensive cell death, accounting for the abundant spherical-like cells (Figure 5B, right image). Overall, the cell viability data in Figure 5 demonstrate significantly more pronounced cell toxicity of Aβ42 induced upon its interactions with BIM-BH3.

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DISCUSSION The structural, functional, and biological data presented in Figures 1−5 point to an intriguing and previously undisclosed mechanism which might account for the extensive neuronal cell death encountered in Alzheimer’s disease. Specifically, our results indicate that the proapoptotic sequence BIM-BH3 experiences affinity and distinct binding with Aβ42 (Figure 1). Interactions between Aβ42 and BIM-BH3, which have not been previously reported, are conceivable in actual cellular environments, as BIM-BH3 is present both in the mitochondria and cytosol,67 while several reports have also localized Aβ42 in these cellular compartments.10−12,68 A critical aspect of Aβ42 structural evolution concerns its aggregation pathways, since Aβ42 oligomers and protofibrillar species are believed to be major cytotoxicity and pathogenicity factors.1 Indeed, the experimental data in Figure 2 demonstrate that BIM-BH3 interactions resulted in significant remodeling of Aβ42 structure and aggregation. Specifically, BIM-BH3 affected the solution conformation of Aβ42, promoting adoption of β structure by the peptide (Figure 3A), which went hand-in-hand with acceleration of aggregation manifested in the ThT fluorescence experiment (Figure 2B). These F



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intertwined phenomena were dramatically visualized in the AFM experiment which revealed ubiquitous smaller Aβ42 fibrils upon incubation with BIM-BH3, in contrast to the longer, mature Aβ42 fibers assembled in solution without the presence of BIM-BH3. Aβ42 oligomers and protofibrils exhibit significant interactions with cellular membranes. Indeed, the fluorescence anisotropy experiments in Figure 4 complemented the spectroscopy and microscopy analyses in Figures 2 and 3, demonstrating that BIM-BH3 induced enhanced Aβ42/ membrane interactions. Notably, in two different membrane models, one mimicking the plasma membrane (Figure 4A) while another designed to mimic the mitochondrial membrane (Figure 4B), preincubation of Aβ42 with BIM-BH3 resulted in experimentally significant “stiffening” of the lipid bilayer, likely reflecting insertion of membrane-active oligomeric species.69 While the experimental data in Figures 1−4 underscore the interactions between Aβ42 and BIM-BH3 and their consequences in solution and in the presence of biomimetic membrane models, a critical question one needs to address is whether such interactions actually impact cell viability and cellular processes. Importantly, flow cytometry and cell morphology analyses depicted in Figure 5 show that BIMBH3/Aβ42 mixtures exerted higher neuronal cell toxicity compared to Aβ42 or BIM-BH3 alone, and that the toxicity effect was dependent upon BIM-BH3 concentration. These results nicely complement the biophysical analyses in Figures 1−4 and suggest that the BH3-induced greater abundance of oligomeric and prefibrillar Aβ42 species gave rise to cell death via enhanced membrane interactions. Figure 6 depicts a proposed model based upon our experimental data presented in Figures 1−5, accounting for the pronounced neurotoxicity associated with BIM-BH3/Aβ42 interactions. The top part of the cartoon illustrates the structural transformation of Aβ42 monomers, produced through the enzymatic activity of APP, into membrane-active oligomers and protofibrils, believed to constitute major toxic determinants. The Aβ42 oligomers ultimately assemble into mature fibrils which accumulate upon the neuronal cells. The model in Figure 6 suggests, however, that coincubation of Aβ42 with BIM-BH3 promotes assembly of membranedisruptive and cytotoxic Aβ42 oligomers, accounting for the enhanced neuronal cell death encountered in Alzheimer’s disease.

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METHODS

Materials. Aβ42 was purchased from AnaSpec in a lyophilized form at >95% purity. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (ovine wool, >98%), sphingomyelin (brain, porcine), and cardiolipin (heart, bovine) were purchased from Avanti Polar Lipids. Hexafluoroisopropanol (HFIP), sodium hydrosulfite, and sodium phosphate monobasic were purchased from SigmaAldrich (Rehovot, Israel). 1,6-Diphenylhexatriene (DPH) was obtained from Molecular Probes, Inc. (Eugene, OR). Synthesis of BIM-BH3. A Liberty microwave peptide synthesizer from CEM was used for synthesis of the BH3 domain of the BIM protein (sequence DMRPEIWIAQELRRIGDEFNAYYARR). Stepwise coupling reactions were performed with Fmoc strategy on a 0.25 mmol scale using Fmoc-Arg(Pbf)-OH Wang resin (0.5 mmol/g, Iris Biotech), enantiomerically pure Fmoc-protected amino acids, 1hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and diisopropylethylamine (DIPEA) (4:4:8 equiv). Each amino acid was coupled using standard coupling conditions (75 °C, 24 W, 300 s). N-Terminal deprotection of the Fmoc group during the syntheses was performed twice using 20% piperidine in DMF (first cycle: 75 °C, 35 W, 30 s; second cycle: 75 °C, 50 W, 180 s). The synthesis results in the linear resin-bound side chain protected Fmoc-Bim-Wang-resin. The FmocBim-Wang-resin was then completely transferred into a filtercontaining syringe for Fmoc deprotection, followed by simultaneous side chain deprotection and cleavage from the resin. The Fmoc deprotection was performed twice using 20% piperidine in DMF (2 mL). After Fmoc deprotection, the resin was washed with DMF (2 × 2 mL) and DCM (2 × 2 mL), shrunk with diethyl ether, and dried under vacuum for 30 min. Finally, cleavage of the Bim peptide from the resin was performed using 1 mL of cleavage mixture containing TFA/H 2 O/triisopropylsilane (TIS)/1,2-ethanedithiol (EDT) (90%:2.5%:2.5%:5%, v/v) for 6 h at room temperature. The resin was filtered and washed with TFA (0.25 mL). Addition of cold diethyl ether/hexane mixture (1:1, v/v) yielded a white precipitate, which was repeatedly washed with diethyl ether/hexane mixture (1:1, v/v). The crude peptide was then dissolved in acetonitrile/water (1:1, v/v) and lyophilized. The Bim peptide was purified by RP-HPLC using a RP-C18 semipreparative (Varian Dynamax, 21.4 mm × 250 mm) columns on a Varian Prostar Instrument using a gradient of acetonitrile/water containing 0.1% TFA (40 min, 2.5 mL/min; 0 min: 5% ACN, 5 min: 5% ACN; 25 min: 95% ACN, 30 min: 95% ACN, 35 min: 5% ACN, 40 min: 5% ACN). After collecting the desired fraction from HPLC, the purity of the peptide was confirmed using a RP-C8 analytical HPLC column (Varian Dynamax, 4.6 mm × 250 mm). Peptide and Sample Preparations. Aβ42 was dissolved in HFIP at a concentration of 1 mg/mL and stored at −20 °C until use to prevent aggregation. For each experiment, the solution was thawed, and the required amount was dried by evaporation for 6−7 h to remove the HFIP. The dried peptide sample was dissolved in 10 mM sodium phosphate, pH 7.4, at room temperature. Stock solutions of Bim peptide were prepared at 0.5 mM in deionized water and diluted into the Aβ42 solutions at the required concentrations. Vesicle Preparation. Vesicles consisting of DOPC/cholesterol/ sphingomyelin and DOPC/cardiolipin were prepared by dissolving the lipid components in chloroform/ethanol (1:1, v/v) and drying together in vacuum. Small unilamellar vesicles (SUVs; DOPC/Cho/ Sph and DOPC/CL, 0.67:0.25:0.08 and 0.96:0.04 molar ratio, respectively) were prepared in 10 mM sodium phosphate (pH 7.4) by probe sonication (power: 130 W, frequency: 20 kHz, at 20% amplitude) of the aqueous lipid mixtures at room temperature for 10 min. Vesicle suspensions were allowed for 1 h at room temperature prior to usage. Surface Plasmon Resonance (SPR). The constant for binding of Aβ42 to BIM-BH3 peptide was determined by SPR using a ProteOn XPR36 instrument (Bio-Rad) as follows. Aβ42 was immobilized on the surface of the GLC chip by using the amine coupling reagents sulfo-NHS (5 mM N-hydroxysuccinimide) and EDC (20 mM 1-ethyl-

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CONCLUSIONS In conclusion, this study discloses an unrecognized mechanism, according to which interactions between the apoptotic BIM protein interacts and beta-amyloid may account for the devastating neurotoxic effects of Alzheimer’s disease. The model reported here may open new therapeutic avenues targeting BIM/Aβ42 interactions as a possible remedy for Alzheimer’s disease. Notably, elevated BIM levels have been observed in other amyloidogenic pathologies including type-II diabetes28 and Huntington’s disease,30 hinting that the phenomenon we report might be a more universal toxicity determinant in protein misfolding diseases. It is important to note, however, that AD is a complex disease likely affected by contributions from multiple molecular pathways. Thus, it should be emphasized that the precise physiological roles of BIM/Aβ42 interactions and their relationships with AD pathogenesis require further studies. G


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Flow Cytometry for Apoptosis Analysis. Aβ42 in the absence or presence of different BIM-BH3 concentration were dissolved at 37 °C in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) and incubated overnight before being added to each well. SH-SY5Y cells were seeded at 2 × 105 cells per well in 96-well flat plates and were allowed to adhere overnight, followed by incubation with medium containing Aβ42 and BIM samples for additional 48 h. Control cells were incubated with medium that was treated in the same manner with or without BIM peptide. For FACS analysis, the adherent cells were trypsinized, detached, and combined with floating cells from the original growth medium. Cells were washed in Ca2+ containing binding-buffer and stained with Annexin V−APC (Biolegend cat# 561012) for 15 min at room temperature and then resuspended in 150 μL of binding buffer and propidium iodide (PI) for flow cytometer readings. Data from at least 104 cells were acquired using a Gallios flow cytometer (Beckman Coulter) and were analyzed using Kaluza software (Beckman Coulter, version 1.5). Results are presented as means ± SEM. Each experiment was repeated three times. Dot Blot Assay. Oligomers of Aβ42 were prepared in the absence or presence of BIM-BH3 and probed by the oligomer-specific polyclonal antibody (pAb) A11 using a modification of previously described methods. Briefly, HFIP-treated Aβ42 was dissolved in 60 mM NaOH at 2 mM. This solution was sonicated for 1 min followed by dilution with 10 mM phosphate buffer saline (PBS) to a final peptide concentration of 45 μM. The resulting solution was maintained at room temperature without agitation up to 1 h. Periodically, 2 μL aliquots were applied to nitrocellulose membranes. The membranes were blocked for 1 h with 5% nonfat milk in 10 mM Tris-buffered saline (TBS) followed by incubation with A11 at 1:1000 dilution in TBS containing 5% nonfat milk followed by appropriate horseradish peroxidase-linked secondary polyclonal antibodies and developed using an enhanced chemiluminescence (ECL) reagent kit (GE Healthcare).

3-(3-(dimethylamino)propyl)-carbodiimide, Bio-Rad). The Aβ42 protein (2 μg) was immobilized on the chip in 10 mM sodium acetate buffer (pH 4) and run at 30 μL/s for 300 s. BSA (3 μg) was immobilized on the chip as a negative control. Unbound carboxyl residues were deactivated with 1 M ethanolamine HCl at pH 8.5. Each binding assay, were done using PBST (PBSx1 + 0.005% Tween 20) at 25 °C. BIM peptide (the analyte) was then allowed to flow over the surface-bound at five different concentrations (20, 10, 5, 2.5, 1.25, and 0 μM) and a flow rate of 25 μL/min. The next step was to examine the dissociation of the proteins while allowing PBST to flow over the surface for 1800 s. After each run, a regeneration step was performed with 50 mM NaOH at a flow rate of 100 μL/min. BSA reference channel and the BIM-BH3 zero concentration were used as background and subtracted from the BIM sensograms. Data was analyzed with the proteOn manager software version 3.01 using the equilibrium model for measuring affinity. Thioflavin T (ThT) Fluorescence Assay. ThT fluorescence measurements were conducted at 37 °C using 96-well path cell culture plates on a Varioskan plate reader (Thermo, Finland). Measurements were made on samples containing 10 μM Aβ42 in the absence or presence of different BIM-BH3 concentrations. A 180 μL aliquot of the aggregation reaction was mixed with 20 μL of 100 μM ThT in sodium phosphate buffer, pH 7.4. The device was programmed to record fluorescence intensity every 5 min for 14 h. The fluorescence intensity was measured following a 10 min incubation at λex = 440 and λem = 485 nm. Transmission Electron Microscopy (TEM). Peptide aliquots (5 μL) from samples used in the ThT experiments (after 14 h incubation) were placed on 400-mesh copper grids covered with a carbon-stabilized Formvar film. Excess solutions were removed following 2 min of incubation, and the grids were negatively stained for 30 s with a 1% uranyl acetate solution. Samples were viewed in an FEI Tecnai 12 TWIN TEM instrument operating at 120 kV. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded in the range of 190−260 nm at room temperature on a Jasco J-715 spectropolarimeter, using 1 mm quartz cuvettes. Solutions composed of 400 μL contained 25 μM Aβ42 in the absence or presence of different BIM-BH3 peptide concentration. Spectra were recorded at time 0 and 30 min and after 12 h incubation. CD signals resulting from buffer and Bim peptide were subtracted from the corresponding spectra. Atomic Force Microscopy (AFM). Peptide samples (Aβ42 or Aβ42/BIM-BH3 mixtures solution) were scanned by AFM (Dimension 3100 SPM, Digital Instruments Veeco, NY), in tapping mode, following deposition of 5 μL peptide solution (25 μM peptide concentration) in the absence or presence of lipid vesicles (final concentration 0.3 mM) on a freshly cleaved mica substrate. After 5 min, 100 μL of 10 mM phosphate buffer, pH 7.4, was added and the sample was imaged under aqueous solution. All images were acquired using a silicon probe (AC 240, Olympus) with a spring constant of 2 N/m, frequency 70 kHz and a tip with a radius of 9 nm. Fluorescence Anisotropy. The fluorescence probe DPH was incorporated into the SUVs (DOPC/Chol/Sph and DOPC/CL, 0.67:0.25:0.08 and 0.95:0.05 mol ratio, respectively) by adding the dye dissolved in THF (1 mM) to vesicles up to a final concentration of 1.25 μM. After 30 min of incubation at 30 °C of DPH, fluorescence anisotropy was measured at λex = 360 nm and λem = 430 nm using a FL920 spectrofluorimeter (Edinburgh Co., Edinburgh, UK). Data were collected before and after the addition of freshly dissolved Aβ42, BIM-BH3, or their mixture solution. Anisotropy values were automatically calculated by the spectrofluorimeter software using the equation: r = (IVV − GIVH)/(IVV + 2GIVH), G = IHV/IHH, in which IVV is with excitation and emission polarizers mounted vertically; IHH corresponds to the excitation and emission polarizers mounted horizontally; IHV is the excitation polarizer horizontal and the emission polarizer vertical; IVH requires the excitation polarizer vertical and emission polarizer horizontal. Each experiment was repeated at least three times. Results are presented as means ± standard error of the mean (SEM) of seven replicates. *p < 0.001 compared to control vesicles of the particular experiment.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.9b00177. TEM images, dot blot assay, and AFM images (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Stanislav Engel: 0000-0001-5916-5190 Nils Metzler-Nolte: 0000-0001-8111-9959 Raz Jelinek: 0000-0002-0336-1384 Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AD, Alzheimer’s disease; SPR, surface plasmon resonance; AFM, atomic force microscopy; CD, circular dichroism; REFERENCES

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