Alzheimer\xe2\x80\x99s A\xce\xb242 and A\xce\xb240 form mixed oligomers with direct molecular interactions
Formation of A\xce\xb2 oligomers and fibrils plays a central role in the pathogenesis of Alzheimer\xe2\x80\x99s disease. There are two major forms of A\xce\xb2 in the brain: A\xce\xb242 and A\xce\xb240. A\xce\xb242 is the major component of the amyloid plaques, but the overall abundance of A\xce\xb240 is several times that of A\xce\xb242. In vitro experiments show that A\xce\xb242 and A\xce\xb240 affect each other\xe2\x80\x99s aggregation. In mouse models of Alzheimer\xe2\x80\x99s disease, overexpression of A\xce\xb240 has been shown to reduce the plaque pathology, suggesting that A\xce\xb242 and A\xce\xb240 also interact in vivo. Here we address the question of whether A\xce\xb242 and A\xce\xb240 interact with each other in the formation of oligomers using electron paramagnetic resonance (EPR) spectroscopy. When the A\xce\xb242 oligomers were formed using only spin-labeled A\xce\xb242, the dipolar interaction between spin labels that are within 20 \xc3\x85 range broadened the EPR spectrum and reduced its amplitude. Oligomers formed with a mixture of spin-labeled A\xce\xb242 and wild-type A\xce\xb242 gave an EPR spectrum with higher amplitude due to weakened spin-spin interactions, suggesting molecular mixing of labeled and wild-type A\xce\xb242. When spin-labeled A\xce\xb242 and wild-type A\xce\xb240 were mixed to form oligomers, the resulting EPR spectrum also showed reduced amplitude, suggesting that wild-type A\xce\xb240 can also form oligomers with spin-labeled A\xce\xb242. Therefore, our results suggest that A\xce\xb242 and A\xce\xb240 form mixed oligomers with direct molecular interactions. Our results point to the importance of investigating A\xce\xb242-A\xce\xb240 interactions in the brain for a complete understanding of Alzheimer\xe2\x80\x99s pathogenesis and therapeutic interventions.
INTRODUCTION<!>Preparation of wild-type A\xce\xb2 proteins.<!>Preparation of spin-labeled A\xce\xb242.<!>Preparation of A\xce\xb2 globulomers.<!>Transmission electron microscopy.<!>EPR spectroscopy.<!>RESULTS AND DISCUSSION
<p>Deposition of AÎ² aggregates plays a central role in the pathogenesis of Alzheimerâs disease [1]. AÎ² is produced from the proteolytic digestion of amyloid precursor protein by Î²- and Î³-secretases [2]. The cleavage by Î³-secretase is not specific, generating a number of AÎ² proteins with various C-terminal ends. The most abundant AÎ² species is the 40-residue AÎ²40, followed by the 42-residue AÎ²42. Although AÎ²40 is several fold more abundant than AÎ²42 in the brain [3â5], the main AÎ² species in amyloid plaques is AÎ²42 [6â8]. Several studies have shown that AÎ²40 is present only in a subset of amyloid plaques [6,7,9], suggesting that AÎ²42 aggregation may precede AÎ²40 aggregation. Overexpression of AÎ²40 in mouse models of Alzheimerâs disease reduced the plaque pathology, suggesting a protective role for AÎ²40 [10]. Therefore, studying interactions between AÎ²42 and AÎ²40 may provide mechanistic insight into the pathogenesis of Alzheimerâs disease and facilitate development of therapeutic interventions.</p><p>In vitro studies show that AÎ²42 and AÎ²40 interact at molecular level and affect each otherâs aggregation. Surface plasmon resonance experiments showed direct interaction between AÎ²40 and AÎ²42 monomers [11]. NMR studies suggest that AÎ²40 inhibits the aggregation of AÎ²42 by binding to existing AÎ²42 aggregates [12]. Jan et al. [13] showed increasing AÎ²40/AÎ²42 ratio reduced fibril formation and led to reduced toxicity. The fibrils of AÎ²40 can seed the aggregation of AÎ²42, and vice versa [14,15]. Our previous EPR studies show that AÎ²42 and AÎ²40 form interlaced amyloid fibrils [16]. A recent NMR study found that mixing of AÎ²40 and AÎ²42 led to the formation of a fibril whose structure is different from that of either pure AÎ²40 or AÎ²42 fibrils [17].</p><p>In addition to amyloid fibrils, AÎ² aggregation also generates soluble oligomers, which are considered to be the more toxic and pathogenic form of AÎ² aggregates [18]. Using an environment-sensitive fluorophore, Frost et al. [19] showed that AÎ²40 and AÎ²42 formed mixed pre-fibrillar aggregates. Iljina et al. [20] used single-molecule two-color coincidence detection to show that AÎ²42 and AÎ²40 co-oligomerize at physiologically relevant concentrations. More recently, Baldassarre [21] used Fourier transform infrared spectroscopy to show that AÎ²42 and AÎ²40 form mixed oligomers with randomly distributed AÎ²42 and AÎ²40 strands in the Î²-sheet. In all three studies, the oligomers were prepared by lowering the sample pH from basic to neutral. Specific oligomer protocols such as AÎ²-derived diffusible ligands [22] and globulomers [23] are used to prepare oligomers for in vitro [24,25] and in vivo studies [18,26,27]. But no studies have been reported on AÎ²40-AÎ²42 interactions in these specific oligomer preparations.</p><p>In this work, we aim to study whether AÎ²42 and AÎ²40 directly interact with each other in AÎ² globulomers, which are prepared in the presence of low concentrations of SDS [23,28]. The continuous-wave EPR spectrum is sensitive to spin-spin interactions when spin labels are within a range of approximately 20 â«. In a previous study of AÎ²42 globulomers [29], we showed that the spin labels introduced at the same residue position are approximately 10â15 â« apart, consistent with an antiparallel Î²-sheet arrangement in globulomers. The EPR spectra of AÎ²42 oligomers formed by 100% spin-labeled AÎ²42 are broadened by spin-spin interactions, which are diminished in globulomers formed by a mixture of spin-labeled and wild-type AÎ²42. This provides us an approach to study the interactions between AÎ²42 and AÎ²40 in the globulomers (Figure 1). Globulomers prepared with a mixture of spin-labeled AÎ²42 and wild-type AÎ²40 will show reduced spin-spin interactions if AÎ²42 and AÎ²40 interact at molecular level, and unchanged spin-spin interactions if AÎ²42 and AÎ²40 do not interact at molecular level.</p><!><p>Detailed procedure of AÎ² preparation has been described previously [29]. Briefly, AÎ²40 and AÎ²42 proteins were expressed in E. coli C41(DE3) cells (Lucigen) using a fusion protein construct, GroES-ubiquitin-AÎ² [30]. Following purification using a nickel column under denaturing conditions, the fusion protein partners were cleaved off using a deubiquitylating enzyme, Usp2-cc [31]. This leads to the purification of full-length AÎ²40 or AÎ²42 proteins without any extra residues. Finally, AÎ² proteins were buffer exchanged to 30 mM ammonium acetate, pH 10, lyophilized, and stored at â80Â°C. Throughout the purification process, AÎ² proteins were kept in buffers of pH 10 and never exposed to acidic conditions. The identity of the protein was confirmed using mass spectrometry.</p><!><p>A cysteine mutant of AÎ²42, G37C, was constructed using site-directed mutagenesis approach with QuikChange mutagenesis kit (Agilent). Protein purification was performed similarly as wild-type AÎ². For spin labeling, AÎ²42 G37C protein was first treated with 10 mM TCEP at room temperature for 20 min to break any disulfide bonds. Then the protein was buffer exchanged to labeling buffer (20 mM MOPS, 7 M guanidine hydrochloride, pH 6.8). Spin labeling reagent MTSSL, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate (Adipogen), was added at 10-fold molar excess and incubated at room temperature for 1 h. Then the labeling mixture was buffered exchanged to 30 mM ammonium acetate (pH 10), lyophilized, and stored at â80Â°C. The extent of labeling was evaluated with mass spectrometry. Only samples with labeling efficiency of >95% were used in the subsequent experiments. The spin label side chain is named R1 and the spin-labeled AÎ²42 mutant is thus named G37R1.</p><!><p>Lyophilized powder of wild-type AÎ²40, wild-type AÎ²42, and spin-labeled AÎ²42 (G37R1) was first dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 1 mM, bath-sonicated for 5 min, and then incubated at room temperature for 30 min. HFIP was allowed to evaporate overnight in a fume hood. HFIP treated samples were solubilized in dimethyl sulfoxide (DMSO) at 5 mM and bath-sonicated for 5 min. The AÎ² concentration was determined using a fluorescamine method [32]. We prepared four samples of AÎ² globulomers. Sample 1 is 100% spin-labeled AÎ²42. Sample 2 is a mixture 25% spin-labeled and 75% wild-type AÎ²42. Sample 3 is a mixture of 25% spin-labeled AÎ²42, 25% wild-type AÎ²42, and 50% wild-type AÎ²40. Sample 4 is a mixture of 25% spin-labeled AÎ²42 and 75% wild-type AÎ²40. To prepare globulomers, these four AÎ² samples in DMSO were diluted using phosphate-buffered saline (PBS) (20 mM phosphate, 140 mM NaCl, pH 7.4) to 400 Î¼M containing 0.2% (final concentration) SDS. After incubation for 6 h at 37Â°C, these four AÎ² samples were further diluted with 3 volumes of deionized water to a final concentration of 100 Î¼M and incubated for another 18 h at 37Â°C. For collecting the globulomers, the samples were centrifuged at 14,000 g for 20 min to remove insoluble aggregates, and the supernatant was concentrated using an ultrafiltration filter with 30-kD molecular mass cut-off. Samples in the retentate contained the globulomers.</p><!><p>AÎ² globulomers (5 Î¼L) was placed on glow-discharged copper grids covered with 400-mesh Formvar/carbon film (Ted Pella). Then the samples were negatively stained using 2% uranyl acetate and examined under a JEOL JEM-1200EX transmission electron microscope at 80 kV.</p><!><p>AÎ² globulomer samples were loaded into glass capillaries (VitroCom) sealed at one end. A Bruker EMX EPR spectrometer equipped with the SR4102ST cavity was used for X-band continuous-wave EPR measurements at room temperature. A microwave power of 20 mW and a modulation frequency of 100 kHz were used. Modulation amplitude was optimized for each individual sample. Scan width was 200 G. All EPR spectra were normalized to the same number of spins.</p><!><p>To investigate whether AÎ²42 and AÎ²40 form mixed oligomers with molecular interactions, we took advantage of the dipolar interaction between spin labels when they are in a range of approximately 20 â«. The dipolar interaction broadens the EPR spectrum. As shown in Figure 1, when AÎ²42 globulomers are prepared with 100% spin-labeled AÎ²42, the resulting EPR spectrum is broader and has lower amplitude than globulomers prepared with a mixture of spin-labeled and wild-type AÎ²42. This is because interdigitation between spin-labeled and wild-type AÎ²42 in the oligomer increases the distance between spin labels, and thus reduces the dipolar spin-spin interaction. If AÎ²40 and AÎ²42 interact with each other at molecular level, then a mixture of spin-labeled AÎ²42 and wild-type AÎ²40 will lead to a similar level of reduction in the dipolar interaction between spin-labeled AÎ²42.</p><p>Using spin-labeled AÎ²42, G37R1, where R1 represents the spin label, wild-type AÎ²42 and AÎ²40, we prepared four globulomer samples with different ratios of the three AÎ² proteins. The first sample is prepared with 100% spin-labeled AÎ²42. Our previous studies show that the spin labels at position 37 are approximately 12 â« apart in the globulomer [29]. This sample gives strong dipolar interaction between spin labels. The second sample is a mixture of 25% spin-labeled AÎ²42 and 75% wild-type AÎ²42. This sample provides a control for reduced dipolar interaction. The third sample is a mixture of 25% spin-labeled AÎ²42, 25% wild-type AÎ²42, and 50% wild-type AÎ²40. If AÎ²40 does not form molecular interactions with AÎ²42, we will observe a reduction of dipolar interaction but less than the mixture of 25% labeled and 75% wild-type AÎ²42 sample. If AÎ²40 interacts with AÎ²42 in the oligomers, then this sample will give an EPR spectrum that is similar to the 75% wild-type AÎ²42 sample. The fourth sample is a mixture of 25% spin-labeled AÎ²42 and 75% wild-type AÎ²40. In the absence of AÎ²40-AÎ²42 interaction, this sample will give an EPR spectrum that is the same as the 100% spin-labeled sample. In the presence of molecular interactions between AÎ²40 and AÎ²42, this sample will resemble the mixture of 25% labeled and 75% wild-type AÎ²42 sample.</p><p>We examined the morphology of the four globulomer preparations using transmission electron microscopy (Figure 2). In addition to stand-alone oligomers (open arrowheads in Figure 2), we found that the AÎ² globulomers have strong tendency to bind to each other, forming elongated and networked structures. Importantly, the four samples show similar morphology under electron microscope, suggesting that mixing of spin-labeled AÎ²42 with either wild-type AÎ²42 or AÎ²40 had little effect on the globulomer morphology.</p><p>Next we performed EPR measurements on these four globulomer samples. As shown in Figure 3A, globulomers of 100% spin-labeled AÎ²42, G37R1, show dipolar-broadened spectrum with smaller amplitude than the EPR spectrum of mixed globulomers of 25% labeled and 75% wild-type AÎ²42. When globulomers are prepared with AÎ²42 and AÎ²40, either in a mixture of 25% spin-labeled AÎ²42, 25% wild-type AÎ²42, and 50% wild-type AÎ²40, or a mixture of 25% spin-labeled AÎ²42 and 75% wild-type AÎ²40, the resulting EPR spectra are extremely similar to the mixture of 25% labeled AÎ²42 and 75% wild-type AÎ²42, suggesting that AÎ²40 can substitute AÎ²42 in forming mixed globulomers.</p><p>Although we observed abundant elongated and networked structures formed by AÎ² globulomers, the EPR spectra consist of sharp lines, suggesting highly mobile structure in the oligomers (Figure 3A). These spectral features are distinct from the EPR spectra of amyloid fibrils formed by the same spin-labeled AÎ²42 mutant (Figure 3B). In the fibrils, the EPR spectral lines are much broader, resulting in greatly reduced spectral amplitude. Another characteristic of the fibril spectrum is that the three EPR resonance lines collapse toward the center line as a result of the spin exchange interaction in the parallel in-register Î²-sheet structure of AÎ² fibrils [33,34]. Therefore, even though the AÎ² globulomers have a strong tendency to bind to each other, the structures remain unchanged and are very different from the structures in fibrils.</p><p>The findings that AÎ²42 and AÎ²40 have molecular interactions in globulomers are consistent with previous reports that AÎ²42 and AÎ²40 form mixed prefibrillar aggregates. Previously, Barth and coworkers [21] used Fourier transform infrared spectroscopy to show that AÎ²42 and AÎ²40 form mixed oligomers with randomly distributed AÎ²42 and AÎ²40 strands in the Î²-sheet. In their study, they prepared AÎ² oligomers by rapidly changing the solution pH from >11 to 7.4. In addition to showing the mixed nature of AÎ² oligomers formed by AÎ²42 and AÎ²40, the infrared spectroscopy also provided evidence for the antiparallel signature of Î²-sheets. Another study by Frost et al. [19] used an environment-sensitive fluorescent label to show that AÎ²42 and AÎ²40 formed mixed prefibrillar oligomers when the buffer pH was adjusted from 10 to 7. Iljina et al. [20] used two-color coincidence detection method to show that AÎ²42 and AÎ²40 formed mixed oligomers at physiologically relevant concentrations. What distinguish this work from previous ones is that we prepared oligomers using a well-established protocol for globulomers [23,28]. A similar protocol was used by Paravastu and coworkers to prepare â150 kDa oligomersâ, which were shown to consist of both out-of-register parallel Î²-sheets and antiparallel Î²-sheets in NMR studies [24]. EPR data in our previous work are consistent with antiparallel Î²-sheet structure [29]. Together with the reports on mixed AÎ²42-AÎ²40 amyloid fibrils [16,17], the evidence is strong for molecular interactions between AÎ²42 and AÎ²40 in the aggregation process and the aggregated products.</p><p>Our findings also have in vivo implications. The composition of amyloid fibrils in the brain has been extensively studied. AÎ²42 has been found to be the major component of parenchymal plaques [6â8], even though AÎ²40 is several-fold more abundant than AÎ²42 in the brain [3â5]. Several studies found that soluble AÎ² aggregates isolated from brains of Alzheimerâs disease patients contains both AÎ²42 and AÎ²40 [35â37]. Wildburger et al. [36] used mass spectrometry to study the AÎ² isoforms in soluble AÎ² oligomers and found that both AÎ²42 and AÎ²40 are present in the soluble aggregates, with AÎ²42 clearly more abundant than AÎ²40. Using end-specific antibodies, Yang et al. [37] also found that the high molecular weight AÎ² oligomers consist of both AÎ²42 and AÎ²40. Mass spectrometry revealed the presence of heterogeneous AÎ²42-AÎ²40 dimers in the brain extracts, suggesting that AÎ²42 and AÎ²40 indeed form mixed oligomers in the brain [35]. In mouse models of Alzheimerâs disease, AÎ²40 has been shown to have strong anti-amyloid effect [10]. Using BRI-AÎ²40 construct to overexpress AÎ²40, Kim et al. [10] showed that two fold increase of AÎ²40 levels in Tg2576 mice reduced AÎ² deposition by ~80% at 11 months and ~50% at 20 months. Selective overexpression of AÎ²42 had the exact opposite effect of plaque pathology [38]. There have yet to be studies on the correlation between AÎ²42/AÎ²40 ratio in oligomers and the severity of Alzheimerâs symptom. Such information would provide invaluable insights into the role of AÎ²42-AÎ²40 interaction in the pathogenesis of Alzheimerâs disease and lay foundations for new avenues of therapeutic intervention.</p>