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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Mol Recognit. Author manuscript; available in PMC Feb 23, 2010.
Published in final edited form as:
PMCID: PMC2827260

Preparation of fluorescently-labeled amyloid-beta peptide assemblies: the effect of fluorophore conjugation on structure and function


Recent research has focused on soluble oligomeric assemblies of the 42 amino acid isoform of the amyloid-beta peptide (Aβ42) as the proximal cause of neuronal injury, synaptic loss, and the eventual dementia associated with Alzheimer’s disease (AD). While neurotoxicity, neuroinflammation, and deficits in behavior and memory have all been attributed to oligomeric Aβ42, the specific roles for this assembly in the cellular neuropathology of AD remain poorly understood. In particular, lack of reliable and well-characterized forms of easily detectable Aβ42 oligomers has hindered study of the cellular trafficking of exogenous Aβ42 by neurons in vitro and in vivo. Therefore, the objective of this study is to fluorescently label soluble oligomeric Aβ42 without altering the structure or function of this assembly. Previous studies have demonstrated the advantages of using tapping mode atomic force microscopy (AFM) to characterize the structural assemblies formed by synthetic Aβ42 under specific solution conditions (e.g., oligomers, protofibrils, and fibrils). Here, we extend these methods to establish a strategy for fluorescent labeling of oligomeric Aβ42 assemblies that are structurally comparable to unlabeled oligomeric Aβ42. To compare function, we demon-strate that the uptake of labeled and unlabeled oligomeric Aβ42 by neurons in vitro is similar. AFM-characterized fluorophore-Aβ42 oligomers are an exciting new reagent for use in a variety of studies designed to elucidate critical cellular and molecular mechanisms underlying the functions of this Aβ42 assembly form in AD.

Keywords: AFM, amyloid-beta, fluorescence, assembly, oligomer, fibril, cellular uptake, Neuro-2A cells


Intro to Aβ structure and function

The “amyloid hypothesis” postulates that toxicity resides in the amyloid plaques (composed of Aβ peptides) that develop in the extracellular space in the brains of Alzheimer’s disease (AD) patients. Although extracellular amyloid plaques are a pathological hallmark of AD, soluble oligomeric assemblies of Aβ are most closely associated with the cognitive deficits characteristic of the disease. Thus, AD research has focused on soluble oligomeric assemblies of Aβ42 as the proximal cause of in vitro neurotoxicity, ex vivo loss of neuroplasticity, and in vivo deficits in behavior and memory in various experimental paradigms (Yankner and Lu, 2008). However, the specific roles of oligomeric Aβ42 in the cellular neuropathology of AD remain poorly understood. In particular, cellular trafficking of exogenous oligomeric Aβ42 by neurons in vitro and in vivo is critical to our understanding of mechanisms underlying its neurotoxicity. Studies of this cellular processing have been hindered by the lack of reliable and well-characterized forms of easily detectable Aβ42 oligomers.

Synthetic and recombinant preparations of pure Aβ42 peptide are used to understand the aggregation kinetics and behavior of the peptide, as well as to address Aβ structure and function. Currently, Aβ oligomers are defined by individual investigators using numerous methods, including neurotoxic activities, isolation technique (primarily size exclusion chromatography), size estimation by SDS or native PAGE, several imaging techniques, and reactivity with various Aβ conformation-specific antibodies (for review, (Rahimi et al., 2008)). These studies reveal how critically dependent peptide structure is on the preparation conditions, and importantly, the close relationship between the function and structure of Aβ. These preparations have been used extensively to demonstrate significant functional differences between Aβ42 oligomers and fibrils in a variety of experimental models (Dahlgren et al., 2002; Manelli et al., 2004; Trommer et al., 2005; White et al., 2005; Manelli et al., 2007).

AFM for studying Aβ42 assembly

Numerous studies have demonstrated several advantages of tapping mode AFM for Aβ42 morphological characterization (Roher et al., 1996; Harper et al., 1997; Lambert et al., 1998; Harper et al., 1999; Huang et al., 2000; Legleiter et al., 2004; Mastrangelo et al., 2006) and its folding and interactions with other proteins and membrane models (Yang et al., 1999; Yip et al., 2002; Chaney et al., 2005; McAllister et al., 2005; Choucair et al., 2007; Ha et al., 2007). AFM is particularly well suited to the analysis of peptides and proteins that assemble into a variety of morphologically discrete species, specifically, those like Aβ. AFM is one of the few techniques that provides high-resolution three-dimensional morphological images of the full range of structures formed in a single scan. In addition, in situ AFM studies demonstrate its capabilities for monitoring real-time aggregation kinetics of Aβ (Goldsbury et al., 1999; Kowalewski and Holtzman, 1999; Parbhu et al., 2002; Legleiter and Kowalewski, 2004; Cheng et al., 2007). In our previous studies, AFM has proven very useful in developing conditions that consistently produce homogenous preparations of oligomeric or fibrillar assemblies of Aβ42 (Dahlgren et al., 2002; Stine et al., 2003).

Fluorescent labeling of Aβ42

As researchers become increasingly conscientious about utilizing structurally uniform, well-characterized Aβ preparations, the same criteria must be applied to derivitized Aβ assemblies such as fluorophore-labeled Aβ, prior to their widespread use as experimental tools. The sensitivity for detecting fluorophore-labeled Aβ facilitates a range of experimental approaches. Recent studies are using fluorophore-labeled Aβ42 to elucidate mechanisms underlying the functional ability of microglia to bind and/or internalize and degrade Aβ42 as a pathway contributing to Aβ clearance from the brain extracellular space (Paresce et al., 1996; Chung et al., 1999; Brazil et al., 2000; Li et al., 2000; El Khoury et al., 2007; Majumdar et al., 2007; Giunta et al., 2008; Hickman et al., 2008; Jiang et al., 2008; Parvathy et al., 2008) as well as Aβ42 cellular binding, uptake and toxicity (Clifford et al., 2007; Saavedra et al., 2007; Simakova and Arispe, 2007; Chafekar et al., 2008). Others have used fluorescent Aβ to examine Aβ-induced macrophage activation (Smits et al., 2001) and transport/efflux mechanisms for blood-brain barrier Aβ clearance (Kuhnke et al., 2007). In vitro, trafficking and processing of fluorescent oligomeric Aβ42 by neural cells could be investigated using substantially lower concentrations of Aβ, thus working in the absence of acute neurotoxicity that often accompanies concentrations necessary for subsequent analysis. However, nearly all of the fluorescent Aβ42 preparations used to date originate from different preparation/solubilization methods of labeled or unlabeled Aβ peptide, and in many cases the peptide assemblies have not yet been structurally/morphologically characterized. Thus, structural comparisons between the unlabeled and labeled Aβ assemblies are not possible. Establishing the specific structural form of the assemblies, by AFM and other methods, is necessary to reliably interpret and compare results from the various fluorescent Aβ42 species.

Studies using fluorescent Aβ42 are facilitated by an assortment of amine-reactive fluorescent dyes and a number of N-terminally fluorophore-labeled Aβ42 peptides that are commercially available (Figure 1A shows a selection of those used with Aβ to date). This generates the possibility for diverse and complementary approaches, but also the risk for variability, and inconsistency. For example, properties of specific fluorophores may provide unique information (such as their use as environment sensitive probes). However, as Aβ peptide behavior and function are highly conformation dependent, different fluorophore molecular structures such as charged groups and size may exert different effects on Aβ assembly and function.

Figure 1Figure 1
Fluorescent labeling of Aβ42. (A) Structures of fluorophores commercially available as N-terminal Aβ42 peptide conjugates and as activated reagents for conjugation that have been used in studies on Aβ42. (B) Schematic of two approaches ...

The schematic in Figure 1B outlines two basic strategies for obtaining fluorophore-labeled Aβ42 preparations under conditions previously characterized to generate distinct, uniform assemblies of Aβ oligomers and fibrils. Figure 1C summarizes considerations for each approach. In strategy A, although N-terminally labeled peptides are conveniently available from multiple commercial sources, the effect of the fluorophore on the assembly is likely variable across dyes with uncertain consequences on the final structure and/or multimer stoichiometry. In strategy B, performing the fluorophore conjugation on a stable or quasi-stable Aβ assembly state is straightforward, and not disruptive to the assembly process. However, an unknown degree-of-labeling requires additional experimental determination, and yield and cost may prohibit use in applications requiring high concentrations or quantity.

In this work, we examine these two strategies for preparing labeled Aβ assemblies, evaluating structural effects using AFM. Based on our observations, the presence of an N-terminal fluorophore generates atypical oligomer and fibril formation, whereas labeling stably formed Aβ assemblies leads to fluorescently labeled preparations with unperturbed structural morphology. We characterized our preparation of Alexa Fluor488®-labeled Aβ42 oligomers using AFM and a neuronal cell uptake assay, to demonstrate that their structural morphology and functional behavior is similar to unlabeled Aβ42 oligomers. Therefore, we have developed a protocol to fluorescently label and characterize assembled Aβ42 oligomers as a new tool for structural and functional studies.



Synthetic human amyloid-beta 1–42 was purchased from California Peptide (cat. # 641-15). For experiments comparing unlabeled and N-terminally labeled Aβ42 assemblies, synthetic human amyloid-beta 1–42, HilyteFluor™488-labeled- and FAM-labeled-amyloid-beta 1–42 peptides were purchased from Anaspec (cat. # 24224, 60479-01, 23526-01, respectively). 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) and anhydrous dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (cat. # 52512 and D-2650). Mica sheets and 12 mm stainless steel pucks were from Ted Pella (product # 52 and 16208, respectively). AFM probes were Al-coated Si cantilevers (42 N/m spring constant; ~300 kHz resonance frequency; tetrahedral tip with 7 nm radius (Olympus cat. # OMCL-AC160TS-W2). 1× phosphate-buffered saline (PBS, 4 mM phosphate, 155 mM NaCl, pH (4°C) 7.5) was prepared from a 10× stock (Invitrogen cat. # 70011). Phenol-red free Ham’s F12 media was purchased from Promocell (Heidelberg, Germany, cat. C-72117) and supplemented with l-glutamine (146 g/L) prior to use. 10 mM hydrochloric acid (HCl) was prepared from a 1 M stock (Sigma cat.# H9892). Mouse monoclonal anti-Aβ antibodies to residues 1–16 (6E10) and 17–24 (4G8) were from Signet/Covance.

Oligomer- and fibril-forming conditions

Aβ42 oligomers and fibrils were prepared according to our previously established protocols (Stine et al., 2003). Briefly, to remove pre-existing aggregates, peptide powders from commercial vendors were pre-treated by dissolving as a 1 mM solution in HFIP. Following evaporation of the HFIP in a fume hood overnight, the resulting peptide film was stored desiccated at −20°C. Immediately prior to use the films were allowed to come to room temperature (RT, ~22°C), solubilized to 5 mM in anhydrous DMSO, sonicated in a bath sonicator (Branson, bath temperature set to 22°C) for 10 min and diluted to 100 µM in the oligomer-forming conditions (phenol-red free Hams F12, pH 7.4; incubation at 4°C for 24 h) or fibril-forming conditions (10 mM HCl, pH 2.1; incubation at 37°C for 24 h).

Preparing fluorescent oligomers and fibrils from N-terminally fluorophore labeled Aβ42

Solutions of oligomers and fibrils were prepared exactly as described above for unlabeled Aβ42, starting from HFIP treated peptide films prepared from the manufacturer provided lyophilized powder of FAM-Aβ42 and HiLyteFluor™488-Aβ42.

Labeling Aβ42 oligomers after assembly

Aβ42 oligomers were prepared from HFIP-treated films as described above for unlabeled peptide, except that 1× PBS was used to make the 100 µM solution instead of F12 media. After 24 h incubation at 4°C, labeling was performed using the Alexa Fluor®488 TFP ester Microscale Labeling Kit (A30006, Invitrogen) essentially according to manufacturer protocol. Briefly, 50 µl of 100 µM Aβ oligomer solution was pH adjusted to pH 9 with 5 µl of 1 M NaHCO3, followed by addition of 4 µl of the H2O solubilized reactive dye. Incubation at RT (~22°C) was performed for 15 min followed by immediate addition of 55 µl of the labeling reaction mixture onto a spin column packed with a 425 µl slurry of Biogel P-6 resin for removal of unincorporated dye. The resulting eluant (pH 7.4) was stored for up to 2 days at 4°C or used immediately for AFM and gel/blot characterization, and cellular uptake experiments.

SDS-PAGE/Western blot characterization of labeled Aβ peptides

Gel electrophoresis and Western blot analysis were performed as described previously (Dahlgren et al., 2002; Stine et al., 2003). Briefly, samples were prepared by dilution into a non-reducing 1× sample buffer (to deliver 50–100 pmoles of peptide per lane) using NuPAGE® 4× LDS sample buffer (Invitrogen; 0.56 M Tris base, 0.423 M TrisHCl, 40% (w/v) glycerol, 8% (w/v) LDS, 2 mM EDTA, 0.075% (w/v) Serva Blue G250, 0.025% (w/v) Phenol Red, pH 8.5) with no heating prior to loading. SDS-PAGE was performed using NuPAGE® 4–12% Bis-Tris 1.0 mm gels in MES running buffer at 90–100 V, 80 mA for 80–90 min. After detection of in-gel fluorescence, Western blot transfer to 0.2 µm poly-vinylidene difluoride (PVDF) membrane was performed at 15 mA for 20–30 min followed by 25 V, 160 mA for 1 h. Transferred fluorescent peptides were again visualized on the membrane, followed by blocking in Tris-buffered saline + 0.0065% (v/v) Tween-20 (TBST) containing 5% (w/v) non-fat dry milk for 30–60 min at RT (~22°C). Primary antibody incubations were overnight at 4°C using mouse monoclonal antibodies 4G8 or a 4G8/6E10 mixture (at a dilution of 1:5000 each). For detection, the membrane was incubated with horseradish peroxidase-conjugated rabbit-anti-mouse IgG (1:10 000), developed using enhanced chemiluminescence (ECL) Western blotting substrate (Pierce), and visualized on the Kodak 4000R imaging system. SeeBlue®Plus2 Pre-Stained Standards (Invitrogen) were included for molecular weight estimation. In-gel and on-membrane fluorescence of labeled Aβ was visualized by excitation with ultraviolet epi-illumination (no filters) on the Kodak 4000R system. Alternatively, fluorescence is visualized using the Typhoon 9410 variable mode imager (Amersham Biosciences) in fluorescence mode with the 532 green laser/526 SP emission filter set.

Analysis of SDS-PAGE by Silver Stain was performed according to manufacturer protocol using the Silver Xpress® Silver Staining Kit (Invitrogen).

Atomic force microscopy

Samples were prepared and analyzed by AFM according to previously established methods (Stine et al., 2003). For samples in F12 media, freshly cleaved mica was pre-treated with 3 µl of 1 M HCl and then rinsed with 2 drops 0.02 µm-filtered (Whatman Anotop 10) ultrapure water, followed by immediate addition of 10 µl of sample. For samples in HCl or PBS, 10–20 µl of diluted sample solution was deposited directly onto freshly cleaved mica. Aβ solutions were diluted to 10–30 µM in 0.02 µm-filtered H2O for analysis and incubated for 3 min on mica, washed with 3 drops of 0.02 µm-filtered water and dried with tetrafluoroethane (CleanTex MicroDuster III). Samples were scanned in tapping mode under ambient conditions using a Veeco Multimode with Nanoscope IIIa controller equipped with a MultiMode head using a Vertical Engage EV piezoceramic scanner (Veeco, Santa Barbara, CA). Instrument parameters were optimized for each scan keeping contact force at a minimum, with scan rates between 1–2 Hz, drive amplitude between 20–100 mV (depending on cantilever), amplitude setpoint between 1.4 and 1.5 V. Data were processed to remove vertical offset between scan lines by zero order flattening polynomials using NanoScope Software v. 5.31 R1.

Cellular uptake

Neuro-2A (N2A) mouse neuroblastoma cells (ATCC) were seeded at 30 000 cells/well on poly-d-lysine Cellware 8-well culture slides (BD Biosciences) for 8 h in phenol-red free Dulbecco’s Modified Eagle Medium (DMEM) + 10% (v/v) fetal bovine serum (FBS). Alexa488-labeled (2 µM) or unlabeled (10 µM) synthetic Aβ42 oligomers were added to DMEM supplemented with 1% (v/v) N2 supplement (Invitrogen) and incubated for 16 h at 37°C. At the end of the treatment, cells were washed extensively with PBS and fixed in 4% (v/v) paraformaldehyde for 20 min at RT(~22°C) for imaging. Cells treated with unlabeled Aβ42 oligomers were blocked for 15 min with 3% BSA (w/v), incubated overnight with anti-Aβ (1–16) antibody 6E10 (Covance) at 4°C, followed by 1 h incubation at RT(~22°C) with Alexa488-labeled rabbit-anti-mouse IgG (Invitrogen). Wells were mounted with VectaShield (Vector Labs) fluorescence mounting medium and covered with glass coverslips. Confocal microscopy images were acquired on a Zeiss LSM 510 META, Axiovert 200M laser scanning confocal microscope using a Plan-Apochromate Zeiss 40×/1.3 oil immersion objective. To visualize the 488 nm excited fluorescence from the directly fluorophore-labeled Aβ42 oligomers, or the Alexa488-immunolabeled Aβ42 oligomers, 488 nm laser light (krypton–argon laser), a 488/543 two-notch dichroic excitation mirror, and a 505–530nm bandpass emission filter were used, with optimized photomultiplier tube (PMT) parameters. Cells cultured and treated in parallel slides to those used for confocal imaging were lysed by 15 min incubation in 35 µl RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% SDS, Sigma-Aldrich) containing protease inhibitors (Protease Inhibitor Cocktail Set I, Calbiochem), followed by centrifugation. Super-natants were analyzed for cell-associated Aβ using in-gel fluorescence and western blot detection of SDS-PAGE, with loading volume normalized to total protein concentration as determined by BCA analysis. Samples were heated at 70°C for 10 min in LDS sample buffer containing 5% (v/v) b-mercaptoethanol prior to loading. Fluorescence was visualized on the Typhoon 9410. After transfer, PVDF membrane was incubated for 15 min in boiling 1× PBS prior to blocking and antibody incubations.


We examined the two approaches outlined in Figure 1B to obtain fluorescent Aβ42 oligomer and fibril preparations under well-characterized conditions using three commercially available, spectrally similar fluorescent dyes. To form oligomers and fibrils starting from N-terminally labeled Aβ42 (Strategy A), we used the peptide conjugated to carboxyfluorescein (FAM), and the peptide conjugated to HiLyteFluor™488. For labeling oligomeric and fibrillar Aβ assemblies after their formation (Strategy B), we selected the amine-reactive TFP ester of Alexa Fluor®488 for its increased labeling efficacy, stability in aqueous conditions, and availability in a convenient straightforward labeling kit. Both HilyteFluor™488 and Alexa Fluor®488 are marketed as “next generation” fluorescent dyes that are brighter, more photostable and pH insensitive compared to fluorescein and its derivatives (See Figure 1A for fluorophore structures).

Strategy A: N-terminal fluorophore-labeled Aβ42 forms atypical oligomers and fibrils

We purchased two different N-terminally fluorophore-labeled Aβ42 peptides and performed HFIP treatment to remove pre-existing aggregate seeds according to our standard oligomer and fibril preparation protocols. We found that the specific nature of the N-terminal fluorophore affects subsequent peptide biochemistry. Under oligomer-forming conditions, HiLyte-Fluor™488-labeled Aβ42 remains only partially soluble, while FAM-labeled Aβ42 is soluble. Potential biochemical explanations for the affect of HiLyteFluor488 on Aβ42 solubility are unknown, but may include changes to the fluorophore-peptide conjugate isoelectric point, solution conformation, and/or aggregation such that it is not stable in oligomer-forming conditions (Teplow, 2006). Brightly colored fluorescent Aβ42 solutions of FAM-Aβ42 oligomers and fibrils, and HiLyteFluor™488 fibrils are readily detected as fluorescent bands by UV-excited fluorescence (Figure 2A, left) and as anti-Aβ antibody 4G8 signal by Western blot (Figure 2A, right) following SDS-PAGE. In addition, detection of HiLyte-Aβ42 oligomers is possible by solubilization in LDS sample buffer. A typical SDS-PAGE gel analysis of our Aβ42 preparations includes bands corresponding to the monomer, and a combination of bands for a dimer, trimer, and tetramer (based on molecular weight), and additional high molecular weight smears. The relative intensities of individual bands within this pattern can vary with amount loaded on the gel and method of detection. A few differences between the patterns for unlabeled and N-terminally labeled Aβ oligomer and fibril signals can be observed in Figure 2A. First, unlabeled Aβ trimer and tetramer bands are not readily detected by 4G8 compared to the FAM- and HiLyte-Aβ42. In addition, the smear of high molecular weight signal ranging from approximately 40–100 kDa for unlabeled Aβ42 appears from approximately 35–60 kDa for FAM- and HiLyte-Aβ42. Second, although the two different N-labeled peptides have very similar molecular weight (4873.4 and 4871.5 Da for FAM and Hilyte, respectively, versus 4515.1 Da for unlabeled Aβ), their migration on the SDS-PAGE gel is different. N-terminal FAM-Aβ42 runs just above unlabeled Aβ42 while HiLyte-labeled Aβ42 appears at the same position of unlabeled Aβ42 (Figure 2A). These differences under denaturing SDS-PAGE analysis likely arise from N-terminal fluorophore effects Aβ peptide biochemical properties. Comparison of the left and right panels in Figure 2A reveals that the high molecular weight species appear absent from the fluorescence signal, likely due to fluorescence quenching in highly self-aggregated species. Finally, as demonstrated previously (Dahlgren et al., 2002; Stine et al., 2003), conformation-specific differences between oligomeric and fibrillar preparations of both unlabeled and N-terminally labeled Aβ42 are not readily apparent in this assay (Bitan et al., 2005). Thus, in general, solutions of oligomers and fibrils formed from FAM- and HiLyte-Aβ42 exhibit similar behavior to the unlabeled peptide by SDS-PAGE.

Figure 2
Characterization of Aβ assemblies prepared from N-terminal fluorophore-labeled Aβ42 peptides by SDS-PAGE/Western blot and AFM analysis. (A) Fluorescently labeled Aβ42 preparations appear similar to unlabeled Aβ42 by gel/blot ...

To determine if the presence of the N-terminal fluorophore during oligomer and fibril formation affected the morphology of the assemblies, we characterized N-labeled fluorescent Aβ preparations using AFM to compare unlabeled and N-labeled Aβ42 structural assemblies. Figure 2B shows that the presence of N-terminal FAM on Aβ under oligomer-forming conditions causes a range of variable effects on morphology (Figure 2B, top row). Unlabeled Aβ42 oligomers exhibit the typical appearance of small oligomeric structures in the absence of protofibrils and fibrils. The AFM image of FAM-Aβ42 oligomers includes all types of representative morphologies observed across samples, ranging from round oligomer-like shapes similar to the unlabeled peptide (red arrow), to those exhibiting taller z height (cyan arrow), larger amorphous aggregates (green arrow), and protofibril-like aggregates (grey arrow). Relative abundance of these structures varies among samples. Because HiLyteFluor™488-Aβ42 was only partially soluble under oligomer-forming conditions, AFM analysis was not performed. The bottom row of Figure 2B shows AFM analysis of Aβ42 fibrils. All three types of fibrillar assemblies appear similar in diameter and height with lengths extending over several microns. The unlabeled Aβ42 fibrils exhibit the characteristic curved morphology. However, N-labeled fibrils with both the FAM and HiLyte fluorophores exhibit a straight morphology with amore rigid appearance. It also appears that individual strands tend to align in parallel. In addition, both FAM- and HiLyte-labeled fibril preparations contain short protofibril-like species (red arrow), and increased non-fibrillar small globular Aβ (green arrow) compared to unlabeled fibrils. Interestingly, the morphology of N-terminally fluorophore-labeled fibrils closely resembles that of fibrils formed by a mutant Aβ42 peptide containing the Arctic mutation E22G (Dahlgren et al., 2002). The E22G mutant fibrils also exhibited significantly greater decreases in neuronal viability compared to WT Aβ fibrils. Therefore, changes in fibril and oligomer morphology observed here for N-terminally fluorophore-labeled Aβ assemblies may alter functional properties. These data support that fluorescent Aβ assemblies formed from FAM-Aβ42 and HiLyte-Aβ42 under oligomer- and fibril-forming conditions have structural features distinct from unlabeled oligomers and fibrils.

It may be possible to avoid variable and inconsistent effects of N-labeled peptides on Aβ assembly, such as those observed here for FAM-labeled oligomers, by taking alternative approaches to preparing the Aβ. For example, mixing unlabeled and N-terminally labeled peptide prior to assembly (Chafekar et al., 2008; Nielsen et al., 2008), adding a flexible linker between the fluorophore and the Aβ N-terminus (Frost et al., 2003), or using alternative initial solubilization of the peptide, such as alkaline conditions (Teplow, 2006). However, extensive structural comparisons are needed to assess whether any alternative approach is a suitable option for preparing defined fluorescent Aβ assemblies from N-terminally labeled starting material.

Strategy B: Aβ42 oligomers labeled after formation appear similar to unlabeled oligomers

We also prepared fluorescent Aβ42 assemblies by performing fluorophore conjugation after incubating the peptide under our defined assembly conditions. Aβ42 oligomers were prepared using our standard protocol starting from HFIP-treated unlabeled Aβ42 peptide film, with one modification: the buffer for oligomer formation was switched to 1× PBS, pH 7.4 to avoid the presence of free amines in the buffer during labeling that would compete with the peptide for the Alexa Fluor®488 TFP ester. Post-labeling reaction purification using a microspin column packed with 6 kDa size-exclusion resin removes most of the unincorporated fluorophore dye, yielding a yellow-green solution of brightly fluorescent Aβ42 oligomers. This process also results in sample dilution and loss of material to the beads, with estimated yields of approximately 40%. The purified Alexa Fluor®488-oligomer solution contains a mixture of both unlabeled and labeled Aβ42 peptides, including both singly and doubly-labeled Aβ42 (based on preliminary matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, data not shown).

As shown in Figure 3A, solutions of Alexa Fluor®488-labeled Aβ42 oligomers are readily detected using in-gel fluorescence (left), 4G8-western blot (middle), and silver stain (right). In the left panel, the fluorescent band at the position of Aβ42 confirms successful covalent conjugation of Alexa Fluor®488 to Aβ42 in the oligomer–containing solution. The three-band pattern typically observed for unlabeled Aβ42 (monomer, trimer, tetramer) is also observed for Alexa488-Aβ42 detected by in-gel fluorescence and Silver stain, resembling the banding pattern of unlabeled Aβ42, with the 4G8-detected band at the trimer position very faint for Alexa488-Aβ42. Silver stain also reveals a very faint band between the monomer and trimer bands for both types of Aβ that is positioned higher for unlabeled Aβ being higher than for Alexa Fluor®488-labeled Aβ. Similar to the N-terminally labeled Aβ42 peptides, the conjugation to Alexa Fluor®488 appears to affect certain Aβ42 peptide biochemical properties, reflected in minor differences seen by SDS-PAGE analysis. In addition, solutions of unmodified Aβ42 oligomers tolerate acidic conditions, while Alexa Fluor®488-labeled Aβ42 oligomers exhibit insolubility when the solution pH is lowered to 4 or below. Independent silver stain analysis was also performed to assess the ability of the antibodies (6E10/4G8) to detect the total Aβ present in the preparation (unlabeled, N-labeled and internally-labeled). A partially resolved band atop the abundant signal at the monomer position is observable depending on the detection method (see arrow in the fluorescent and silver stained gels, Figure 3A). This is potentially a different species with altered migration due to the presence of multiple Alexa488 labels on the same peptide, or anomalous migration of the same MW product (similar to observations for FAM vs. HiLyte) due to a single Alexa488 present at a different position than the major product (e.g., at either one of the two internal lysine positions (K16 and K28) instead of the N-terminus).

Figure 3
Characterization of Alexa Fluor®488-labeled Aβ42 oligomers by SDS-PAGE and AFM. (A) Fluorescently labeled Aβ42 oligomers appear similar to unlabeled Aβ42 by gel/blot analysis. Oligomers formed from unlabeled Aβ42 ...

To determine if adding a fluorophore to assembled Aβ42 oligomers affects their morphology, we characterized Alexa Fluor®488-labeled Aβ preparations using AFM. Comparison of their structural morphology to that of unlabeled Aβ oligomer preparations (Figure 3B) shows that both solutions contain small globular oligomers, free from large amorphous aggregates, protofibrils, or fibrils. These data support that the Aβ42 oligomeric assembly state is stable to the labeling procedure and the morphology is unaffected by the fluorophore conjugation. Infrequent observation of short straight fibril-like structures appeared in some samples; however these are minor component. Similar to unlabeled oligomers, Alexa Fluor®488-labeled oligomer solutions can be stored for several days at 4°C or incubated for 24 h at 37°C with negligible changes in the morphology observed by AFM, and extended incubation at 37°C (48 h) leads to eventual appearance of fibril-like structures.

Alexa Fluor® 488-labeled Aβ42 oligomers exhibit similar cellular uptake to unlabeled oligomers

We further characterized Alexa488-labeled oligomers by comparing their function to unlabeled oligomers in a cellular uptake assay. Mechanisms of Aβ42 oligomer internalization by neuronal cells are poorly understood; however receptor-mediated endocytosis by a variety of receptors has been suggested (see (Verdier et al., 2004) for review). As fluorophore labeling may interfere with Aβ42 receptor binding and Aβ42 internalization/uptake could be sensitive to its conformation, evaluation of uptake behavior for fluorophore-labeled Aβ assemblies is an important aspect of their characterization. We compared Aβ uptake using CLSM analysis of immunofluorescence for unlabeled oligomers and direct fluorescence for Alexa Fluor® 488 oligomers (Figure 4, Panel A). The detected fluorescence signals in cells treated with 2 µM Alexa Fluor®488-labeled oligomers (left) and with 10 µM unlabeled Aβ42 (right) both appear as regions of punctate fluorescence, with large extracellular aggregates observed as bright fluorescent spots. In both cases, the majority of cells internalize Aβ, with fluorescent puncta appearing throughout the cell bodies. In addition, antibody-based detection of internalized Aβ appears to label Aβ localized near the perimeter of some cells (right panel); however, this likely arises from the higher concentration of Aβ required for detection by antibody versus directly fluorescently labeled Aβ signal. We also compared cellular uptake by analyzing unlabeled and Alexa Fluor®488-labeled Aβ levels in cell lysates from cells treated in parallel (Figure 4B). Lysates from cells treated with Alexa-labeled Aβ oligomers (top panel) and unlabeled Aβ oligomers (middle panel) both show Aβ signal, confirming cellular uptake of both types of Aβ oligomers. These data suggest that oligomer properties, as measured by cellular uptake, are unperturbed by the post-assembly Alexa Fluor®488 fluorophore modification. Here the advantages of a fluorophore label for direct detection and sensitivity are apparent. Intracellular AlexaFluor®488-labeled Aβ42 arising from cells treated with 2 µM is readily detected by in-gel fluorescence, while the antibody detection fails to pick up this signal. Alexa Fluor®488-labeled oligomer preparations are a mixture of labeled and unlabeled peptide, indicating that the individual Aβ42 subunits comprising each oligomer are not uniformly labeled. This lower degree of labeling may actually be advantageous, as the fluorophore is present at a high enough level for sensitive detection of the oligomers, but, as shown here, not to the extent of interfering with conformation and function.

Figure 4
Following uptake, Alexa Fluor®488-labeled Aβ42 oligomers appear as punctate fluorescence within the cell, similar to unlabeled Aβ42 oligomers. Aβ uptake in Neuro-2A cells was analyzed using confocal laser scanning microscopy ...

A different approach is needed for preparing fluorophore-labeled fibrils

Although strategy B is a suitable approach for labeling oligomers, it cannot be applied to fibrils prepared by our methods. Fibril-forming conditions (pH 1–2) are sub-optimal for aminebased conjugation chemistry. Several modifications to the labeling protocol failed to generate labeled Aβ42 fibrils comparable to unlabeled fibrils. For example, with extended reaction time the poor solubility of the Alexa Fluor®488-TFP ester, and of the resulting fluorophore conjugate at low pH caused the dye and fibril mixture to gel out of solution. Raising the fibril-containing solution pH to a more favorable value for amine conjugation chemistry also proved unsuccessful as the fibril morphology is highly sensitive to pH (Stine et al., 2003). One possible solution for future studies is to form fibrils by extended incubation in PBS (e.g., 1 week) at 37°C and then perform the amine-reactive fluorophore-labeling reaction, similar to Parvathy et al. (2008). However, structural comparison of these fluorescent- fibrils to their well-characterized unlabeled fibrils has not yet been performed.

Comparison to other characterized fluorescent Aβ preparations

Limited characterization of other recently employed fluorophore-labeled Aβ preparations has been performed using various combinations of structural and functional analyses (Webster et al., 2001; Saavedra et al., 2007; Chafekar et al., 2008). In general, within a given experimental analysis technique and Aβ preparation method, at least one feature of unlabeled Aβ is preserved in fluorophore-labeled Aβ preparations. For example, N-terminally fluorescein-labeled oligomers and fibrils prepared using oligomer- and fibril-forming conditions similar to our protocol showed similar morphology to the corresponding unlabeled assemblies by negative-stain transmission electron microscopy (EM) analysis, and their functional characterization revealed similar induction of neuronal apoptosis for labeled and unlabeled preparations (Saavedra et al., 2007). Labeled oligomer and fibril preparations starting from a mixture of 2:1 unlabeled/tetramethylrhodamine labeled-Aβ42 prepared using oligomer-and fibril-forming conditions similar to our protocol also showed comparable morphology to unlabeled assemblies by EM (Chafekar et al., 2008). Functional data showed comparable behavior between unlabeled and labeled Aβ fibrils, but less neurotoxicity for fluorescent-oligomers compared to unlabeled oligomers (Chafekar et al., 2008). In these studies, smaller less obvious changes to structure/morphology may not be readily apparent after fixing for EM, as the oligomers in these images appear clumped together, and height information is not available in these data. Furthermore, when mixtures of labeled and unlabeled peptide are used, it is unknown whether the fluorophore-labeled peptide incorporates into unlabeled assemblies. Taken together, the above results and our data emphasize the importance of characterizing Aβ preparations using multiple complimentary structural and functional techniques, particularly including AFM analyses that enable comparison of detailed morphological features at high resolution.


AFM is an extremely useful tool to monitor possible modifications to Aβ structure/morphology caused by fluorophore-labeling. Our comparison of two strategies for preparing fluorescent Aβ42 assemblies showed that for Aβ oligomers, forming the defined Aβ assemblies prior to fluorophore-labeling offers an approach that preserves the morphology of unlabeled Aβ. On the other hand, the presence of an N-terminal fluorophore during assembly exerts variable effects on Aβ morphology, resulting in formation of atypical oligomers and fibrils. Future studies could further examine how N-terminal fluorophore effects on Aβ peptide structure and biochemical properties relate to the morphological variations observed here, and may reveal additional important details on the role of the N-terminus in structure and function. Alexa Fluor®488-labeled Aβ42 oligomers are directly fluorophore-labeled Aβ assemblies with comparable structure and function to the unlabeled assemblies and will be useful tool to elucidate important structural and functional properties of Aβ42. Future studies are underway to fully characterize the fluorophore/peptide stoichiometry, and the specific location(s) of the fluorophore within individual monomer sequence as well as within the subunits comprising the oligomer assemblies. These studies will also provide useful information on the structural properties of Aβ oligomers. In general, applying AFM structural characterization in parallel to in vitro functional studies allows for observed functions of Aβ to be interpreted in the context of specific assembly states. This is extremely important since a primary question in AD research focuses on identifying the pathogenic Aβ assembly form(s).


The authors gratefully acknowledge financial support for these studies from NIH 1F32AG030256-01 (LMJ), Alzheimer’s Association NIRG-06-26957 (CY), NIH R01 AG19121 (MJL), NIH (NIA) PO1AG021184 (MJL). They also gratefully acknowledge Brian Shy for technical and intellectual contributions, and W. Blaine Stine, PhD for AFM/Aβ training and technical support. Katherine Youmans is acknowledged for assistance with figure preparation.


Amyloid-beta peptide
Alzheimer’s disease
atomic force microscopy
bicinchoninic acid
confocal laser scanning microscopy
lithium dodecyl sulfate
polyacrylamide gel electrophoresis
photomultiplier tube
polyvinyldiene difluoride
Radio-immunoprecipitation assay
room temperature
sodium dodecyl sulfate


  • Bitan G, Fradinger EA, Spring SM, Teplow DB. Neurotoxic protein oligomers–what you see is not always what you get. Amyloid. 2005;12:88–95. [PubMed]
  • Brazil MI, Chung H, Maxfield FR. Effects of incorporation of immunoglobulin G and complement component C1q on uptake and degradation of Alzheimer’s disease amyloid fibrils by microglia. J. Biol. Chem. 2000;275:16941–16947. [PubMed]
  • Chafekar SM, Baas F, Scheper W. Oligomer-specific Abeta toxicity in cell models is mediated by selective uptake. Biochim. Biophys. Acta. 2008;1782:523–531. [PubMed]
  • Chaney MO, Stine WB, Kokjohn TA, Kuo YM, Esh C, Rahman A, Luehrs DC, Schmidt AM, Stern D, Yan SD, Roher AE. RAGE and amyloid beta interactions: atomic force microscopy and molecular modeling. Biochim. Biophys. Acta. 2005;1741:199–205. [PubMed]
  • Cheng IH, Scearce-Levie K, Legleiter J, Palop JJ, Gerstein H, Bien-Ly N, Puolivali J, Lesne S, Ashe KH, Muchowski PJ, Mucke L. Accelerating amyloid-beta fibrillization reduces oligomer levels and functional deficits in Alzheimer disease mouse models. J. Biol. Chem. 2007;282:23818–23828. [PubMed]
  • Choucair A, Chakrapani M, Chakravarthy B, Katsaras J, Johnston LJ. Preferential accumulation of Abeta(1–42) on gel phase domains of lipid bilayers: an AFM and fluorescence study. Biochim. Biophys. Acta. 2007;1768:146–154. [PubMed]
  • Chung H, Brazil MI, Soe TT, Maxfield FR. Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer’s amyloid beta-peptide by microglial cells. J. Biol. Chem. 1999;274:32301–32308. [PubMed]
  • Clifford PM, Zarrabi S, Siu G, Kinsler KJ, Kosciuk MC, Venkataraman V, D’Andrea MR, Dinsmore S, Nagele RG. Abeta peptides can enter the brain through a defective blood-brain barrier and bind selectively to neurons. Brain Res. 2007;1142:223–236. [PubMed]
  • Dahlgren KN, Manelli AM, Stine WB, Jr, Baker LK, Krafft GA, LaDu MJ. Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability. J. Biol. Chem. 2002;277:32046–32053. [PubMed]
  • El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nat. Med. 2007;13:432–438. [PubMed]
  • Frost D, Gorman PM, Yip CM, Chakrabartty A. Co-incorporation of A beta 40 and A beta 42 to form mixed pre-fibrillar aggregates. Eur. J. Biochem. 2003;270:654–663. [PubMed]
  • Giunta B, Zhou Y, Hou H, Rrapo E, Fernandez F, Tan J. HIV-1 TAT inhibits microglial phagocytosis of abeta peptide. Intl. J. Clin. Expmtl. Pathol. 2008;1:260–275. [PMC free article] [PubMed]
  • Goldsbury C, Kistler J, Aebi U, Arvinte T, Cooper GJ. Watching amyloid fibrils grow by time-lapse atomic force microscopy. J. Mol. Biol. 1999;285:33–39. [PubMed]
  • Ha C, Ryu J, Park CB. Metal ions differentially influence the aggregation and deposition of Alzheimer’s beta-amyloid on a solid template. Biochemistry (Mosc) 2007;46:6118–6125. [PubMed]
  • Harper JD, Wong SS, Lieber CM, Lansbury PT. Observation of metastable Abeta amyloid protofibrils by atomic force microscopy. Chem. Biol. 1997;4:119–125. [PubMed]
  • Harper JD, Wong SS, Lieber CM, Lansbury PT., Jr Assembly of A beta amyloid protofibrils: an in vitro model for a possible early event in Alzheimer’s disease. Biochemistry (Mosc) 1999;38:8972–8980. [PubMed]
  • Hickman SE, Allison EK, El Khoury J. Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer’s disease mice. J. Neurosci. 2008;28:8354–8360. [PMC free article] [PubMed]
  • Huang TH, Yang DS, Plaskos NP, Go S, Yip CM, Fraser PE, Chakrabartty A. Structural studies of soluble oligomers of the Alzheimer beta-amyloid peptide. J. Mol. Biol. 2000;297:73–87. [PubMed]
  • Jiang Q, Lee CY, Mandrekar S, Wilkinson B, Cramer P, Zelcer N, Mann K, Lamb B, Willson TM, Collins JL, Richardson JC, Smith JD, Comery TA, Riddell D, Holtzman DM, Tontonoz P, Landreth GT. ApoE promotes the proteolytic degradation of Abeta. Neuron. 2008;58:681–693. [PMC free article] [PubMed]
  • Kowalewski T, Holtzman DM. In situ atomic force microscopy study of Alzheimer’s beta-amyloid peptide on different substrates: new insights into mechanism of beta-sheet formation. Proc. Natl. Acad. Sci. U.S.A. 1999;96:3688–3693. [PMC free article] [PubMed]
  • Kuhnke D, Jedlitschky G, Grube M, Jucker M, Mosyagin I, Cascorbi I, Walker LC, Kroemer HK, Warzok RW, Vogelgesang S. MDR1-P-Glycoprotein (ABCB1) mediates transport of Alzheimer’s amyloid-beta peptides–implications for the mechanisms of Abeta clearance at the blood-brain barrier. Brain Pathol. 2007;17:347–353. [PubMed]
  • Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch DE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1–42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. U.S.A. 1998;95:6448–6453. [PMC free article] [PubMed]
  • Legleiter J, Czilli DL, Gitter B, DeMattos RB, Holtzman DM, Kowalewski T. Effect of different anti-Abeta antibodies on Abeta fibrillogenesis as assessed by atomic force microscopy. J. Mol. Biol. 2004;335:997–1006. [PubMed]
  • Legleiter J, Kowalewski T. Atomic force microscopy of beta-amyloid: static and dynamic studies of nanostructure and its formation. Methods Mol. Biol. 2004;242:349–364. [PubMed]
  • Li R, Shen Y, Yang LB, Lue LF, Finch C, Rogers J. Estrogen enhances uptake of amyloid beta-protein by microglia derived from the human cortex. J. Neurochem. 2000;75:1447–1454. [PubMed]
  • Majumdar A, Chung H, Dolios G, Wang R, Asamoah N, Lobel P, Maxfield FR. Degradation of fibrillar forms of Alzheimer’s amyloid beta-peptide by macrophages. Neurobiol. Aging. 2007;29(5):707–715. [PMC free article] [PubMed]
  • Manelli AM, Bulfinch LC, Sullivan PM, LaDu MJ. Abeta42 neurotoxicity in primary co-cultures: effect of apoE isoform and Abeta conformation. Neurobiol. Aging. 2007;28:1139–1147. [PMC free article] [PubMed]
  • Manelli AM, Stine WB, Van Eldik LJ, LaDu MJ. ApoE and Abeta1–42 interactions: effects of isoform and conformation on structure and function. J. Mol. Neurosci. 2004;23:235–246. [PubMed]
  • Mastrangelo IA, Ahmed M, Sato T, Liu W, Wang C, Hough P, Smith SO. High-resolution atomic force microscopy of soluble Abeta42 oligomers. J. Mol. Biol. 2006;358:106–119. [PubMed]
  • McAllister C, Karymov MA, Kawano Y, Lushnikov AY, Mikheikin A, Uversky VN, Lyubchenko YL. Protein interactions and misfolding analyzed by AFM force spectroscopy. J. Mol. Biol. 2005;354:1028–1042. [PubMed]
  • Nielsen HM, Veerhuis R, Holmqvist B, Janciauskiene S. Binding and uptake of Abeta1–42 by primary human astrocytes in vitro. Glia. 2008 December 5; This article is an Epub ahead of print. Published online: [PubMed]
  • Parbhu A, Lin H, Thimm J, Lal R. Imaging real-time aggregation of amyloid beta protein (1–42) by atomic force microscopy. Peptides. 2002;23:1265–1270. [PubMed]
  • Paresce DM, Ghosh RN, Maxfield FR. Microglial cells internalize aggregates of the Alzheimer’s disease amyloid beta-protein via a scavenger receptor. Neuron. 1996;17:553–565. [PubMed]
  • Parvathy S, Rajadas J, Ryan H, Vaziri S, Anderson L, Murphy GM., Jr Abeta peptide conformation determines uptake and interleukin-1alpha expression by primary microglial cells. Neurobiol. Aging. 2008 March 11; This article is an Epub ahead of print. Published online: [PubMed]
  • Rahimi F, Shanmugam A, Bitan G. Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer’s disease and related disorders. Curr. Alzheimer Res. 2008;5:319–341. [PMC free article] [PubMed]
  • Roher AE, Chaney MO, Kuo YM, Webster SD, Stine WB, Haverkamp LJ, Woods AS, Cotter RJ, Tuohy JM, Krafft GA, Bonnell BS, Emmerling MR. Morphology and toxicity of Abeta-(1–42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer’s disease. J. Biol. Chem. 1996;271:20631–20635. [PubMed]
  • Saavedra L, Mohamed A, Ma V, Kar S, de Chaves EP. Internalization of beta-amyloid peptide by primary neurons in the absence of apolipoprotein E. J. Biol. Chem. 2007;282:35722–35732. [PubMed]
  • Simakova O, Arispe NJ. The cell-selective neurotoxicity of the Alzheimer’s Abeta peptide is determined by surface phosphatidyl-serine and cytosolic ATP levels. Membrane binding is required for Abeta toxicity. J. Neurosci. 2007;27:13719–13729. [PubMed]
  • Smits HA, van Beelen AJ, de Vos NM, Rijsmus A, van der Bruggen T, Verhoef J, van Muiswinkel FL, Nottet HS. Activation of human macrophages by amyloid-beta is attenuated by astrocytes. J. Immunol. 2001;166:6869–6876. [PubMed]
  • Stine WB, Jr, Dahlgren KN, Krafft GK, LaDu MJ. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J. Biol. Chem. 2003;278:11612–11622. [PubMed]
  • Teplow DB. Preparation of amyloid beta-protein for structural and functional studies. Methods Enzymol. 2006;413:20–33. [PubMed]
  • Trommer BL, Shah C, Yun SH, Gamkrelidze G, Pasternak ES, Stine WB, Manelli A, Sullivan P, Pasternak JF, LaDu MJ. ApoE isoform-specific effects on LTP: blockade by oligomeric amyloid-beta1–42. Neurobiol. Dis. 2005;18:75–82. [PubMed]
  • Verdier Y, Zarandi M, Penke B. Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer’s disease. J Pept Sci. 2004;10:229–248. [PubMed]
  • Webster SD, Galvan MD, Ferran E, Garzon-Rodriguez W, Glabe CG, Tenner AJ. Antibody-mediated phagocytosis of the amyloid beta-peptide in microglia is differentially modulated by C1q. J. Immunol. 2001;166:7496–7503. [PubMed]
  • White JA, Manelli AM, Holmberg KH, Van Eldik LJ, LaDu MJ. Differential effects of oligomeric and fibrillar amyloid-beta1–42 on astrocyte-mediated inflammation. Neurobiol. Dis. 2005;18:459–465. [PubMed]
  • Yang DS, Yip CM, Huang TH, Chakrabartty A, Fraser PE. Manipulating the amyloid-beta aggregation pathway with chemical chaperones. J. Biol. Chem. 1999;274:32970–32974. [PubMed]
  • Yankner BA, Lu T. Amyloid beta -protein toxicity and the pathogenesis of Alzheimer’s disease. J. Biol. Chem. 2008;284(8):4755–4759. [PMC free article] [PubMed]
  • Yip CM, Darabie AA, McLaurin J. Abeta42-peptide assembly on lipid bilayers. J. Mol. Biol. 2002;318:97–107. [PubMed]


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