A Microtiter Plate Assay for Polyglutamine Aggregate Extension

Analytical Biochemistry 295, 227–236 (2001) doi:10.1006/abio.2001.5217, available online at http://www.idealibrary.com on

A Microtiter Plate Assay for Polyglutamine Aggregate Extension Valerie Berthelier, J. Bradley Hamilton, Songming Chen, and Ronald Wetzel 1 Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920

Received February 20, 2001; published online July 19, 2001

Polyglutamine (polyGln) aggregates are neuropathological markers of expanded CAG repeat disorders, and may also play a critical role in the development of these diseases. We have established a highly sensitive, fast, reproducible, and specific assay capable of monitoring aggregate-dependent deposition of polyglutamine peptides. This assay allows detailed studies on various aspects of aggregation kinetics, and also makes possible the detection and quantitation of low levels of “extension-competent” aggregates. In the simplest form of this assay, polyGln aggregates are made from chemically synthesized peptides and immobilized onto microplate wells. These wells are incubated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europiumstreptavidin complexation of the immobilized biotin, followed by time-resolved fluorescence detection of the deposited europium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studies on the assembly mechanism of polyGln aggregates and on critical features of the reaction, such as polyGln length dependence. The assay also will be a valuable tool for screening and characterizing antiaggregation inhibitors. It will also be useful for detection and quantitation of aggregation-competent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism. © 2001 Academic Press

Eight inherited neurodegenerative diseases, including Huntington’s disease (HD) 2 and spinocerebellar

ataxia 3, are linked to the expression of an expanded polyglutamine (polyGln) repeat in various unrelated proteins (for reviews see 1, 2). These polyGln expansion-related diseases are progressive disorders characterized by motor and/or cognitive impairments and distinctive pathological patterns of neuronal degeneration. The only mutation implicated in these diseases is an expansion of a polyGln sequence in the diseaserelated protein, generally from a benign length of less than 37 Gln to a pathological length of 38 or more (3–5). All of these neurodegenerative disorders present a common feature: the accumulation of the polyGln repeat disease-related protein into neuronal intranuclear inclusions (6, 7), which have become the neuropathological signature of polyGln disorders (8). The important role that long polyGln repeats play in polyGln-related disorders has been confirmed in a number of models in which mutant forms of various disease proteins were expressed in transgenic mice, Drosophila or Caenorhabditis elegans (9 –15). Although these diseases exhibit similar physiological abnormalities, the only common features of disease-related proteins are the polyGln domains. Because of this, it has been hypothesized that the expanded polyGln is itself responsible for the pathogenesis. Nevertheless, the role of polyGln aggregation in the disease mechanism continues to be controversial (12, 16 – 18). Recent experiments in cell models support the notion that polyGln aggregates are toxic due to their ability to recruit other critical cellular proteins, via their own polyGln components, into the growing aggregate (19 –21). The loss of protein activity due to this sequestration is toxic to the cell. Given the potential role of polyGln aggregates and polyGln aggregate extension in the pathogenesis of

1

To whom correspondence should be addressed at Graduate School of Medicine, R221, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920. Fax: (865) 544-9235. 2 Abbreviations used: HD, Huntington’s disease; polyGln, polyglutamine; TFA, trifluoroacetic acid; HFIP, 1,1,1,3,3-hexafluoro-2-pro0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

panol; RP, reverse phase; IPTG, isopropyl ␤-D-thiogalactopyranoside; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide; ThT, thioflavin T. 227

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expanded CAG repeat diseases, it is essential to characterize the fundamental aggregation behavior of polyGln sequences. Studies of the aggregation behavior dependence on polyGln repeat length are important to fully understand the correlation between length and disease risk, as well as the rules that control the recruitment of other polyGln-containing peptides and proteins into growing polyGln aggregates. In addition, a robust assay for polyGln aggregation and recruitment is required to screen for aggregation inhibitors as potential therapeutics. Furthermore, an assay capable of detecting “extension-competent” or “seeding-competent” aggregates in tissue and serum samples might be crucial for evaluating the role of polyGln aggregates in the disease mechanism. PolyGln sequences are notoriously difficult to work with, and are generally considered to be highly insoluble. To date, the only assay system for assessing polyGln aggregation behavior in vitro is one in which fusion proteins of polyGln-containing fragments of disease-related proteins are expressed in E. coli and proteolytically cleaved to release the polyGln-containing peptide. The aggregation process is subsequently monitored by filtration of SDS-resistant aggregation products, which are revealed by immunostaining (22). Recently, we developed an improved solubilization protocol for chemically synthesized polyGln peptides, for the first time allowing work with long polyGln sequences (23). We describe here a microtiter plate assay for polyGln aggregate formation made possible by this solubilization protocol. This assay, relying on a linked biotin-streptavidin tagging system and timeresolved fluorescence detection of europium, is capable of monitoring polyGln aggregation with sensitivity, reproducibility, and specificity. We also describe results suggesting another configuration of this assay that would allow the detection of very small quantities of seeding-competent polyGln aggregates. MATERIALS AND METHODS

General materials and methods. All peptides were obtained by custom solid-phase syntheses from the Keck Biotechnology Center at Yale University (http: //info.med.yale.edu/wmkeck/). Flanking pairs of Lys residues were included to enhance general solubility by allowing the peptide to be charged in the neutral pH region. PolyGln peptides were obtained in unpurified form and structures and purities confirmed by mass spectrometry at the Keck Center. The biotinylated version of the polyGln peptide was prepared by N-terminal derivatization during the solid-phase synthesis. Solid-phase synthesis of long polyGln tracks can generate significant levels of deletion peptides. For example, MS analysis revealed that the synthetic peptide synthesized as Q 30 was predominantly 29 glutamines

in length, with minor amounts of Q 27, Q 28, and Q 30, giving a weight average repeat length of Q 28. For most of the experiments described here, this heterogeneous product was used in both the biotinylated and the free-amino forms. In one experiment, we purified a sample of biotinyl-K 2Q 30K 2 from the reaction product and tested it in the extension assay. We observed no difference in the extension kinetics between the purified and the slightly heterogeneous forms of the peptide. Solubilization and disaggregation of Q 28 peptides. Disaggregation and solubilization were performed as described (23). Thus, peptides were dissolved in a mixture of 50% trifluoroacetic acid (Pierce, Rockford, IL) and 50% 1,1,1,3,3-hexafluoro-2-propanol (Sigma, St. Louis, MO) (TFA/HFIP) at 0.5 mg/ml, vigorously agitated, and incubated at least 30 min at room temperature. The volatile solvents were evaporated under a stream of argon in a fume hood, and the peptide residue solubilized in H 2O/TFA, pH 3, to a concentration of 0.5 mg/ml. The exact peptide concentration was determined by reverse-phase high-performance liquid chromatography (RP-HPLC) (Hewlett-Packard, Palo Alto, CA) using a Zorbax SB-C3 column. Fifty microliters of solubilized peptides, diluted 10 times in 0.1% TFA, was injected onto the column and analyzed with a 0 –50% (v/v) acetonitrile gradient with 0.05% TFA applied at a rate of 2%/min. The peak area obtained at 215 nm was compared with a standard curve previously established with the peptide K 2Q 15K 2, whose concentration was determined independently by amino acid composition analysis. The HPLC analysis should be conducted immediately after the solubilization step to minimize the waiting time of the disaggregated concentrated peptides. Solutions of peptides should not be stored prior to completing subsequent manipulations as described below. Preparation of polyGln aggregates. K 2Q 28K 2 solubilized as above was adjusted to 10 ␮M (44 ␮g/ml) and the pH raised to 7.4 by addition of a 1/9 vol of a 10X PBS stock. The peptide solution was incubated 24 h at 37°C, followed by 24 h at ⫺20°C. Incubation in the frozen state at ⫺20°C is an important aspect of the preparation of the aggregate (J. B. Hamilton, S. Chen, and R. Wetzel, unpublished); this will be described in detail in a subsequent publication. Thioflavin T fluorescence, light scattering, and electron microscopy were used to characterize the quality of the aggregates (S. Chen and R. Wetzel, manuscript in preparation). Briefly, a 250-␮l aliquot of the reaction mixture is introduced into a fluorescence cuvette and the Rayleigh light scattering assessed in a fluorometer with emission and excitation wavelengths set at 450 nm. Aggregate formation is accompanied by a significant increase in the apparent fluorescence, relative to a solution of

MICROTITER ASSAY FOR POLYGLUTAMINE AGGREGATES

monomeric polyGln in PBS. Thioflavin T is then added to the solution to a final concentration of 50 ␮M, and the fluorescence determined by excitation at 450 nm (5 nm slit) and emission at 482 nm (10 nm slit) (24). Aggregates were collected by centrifugation at 20,800g, 30 min at 4°C, then resuspended to 44 ␮g/ml in extension buffer (PBS 1X, 0.01% Tween 20, and 0.05% NaN 3), and aliquoted into Eppendorf tubes. Tubes were snap-frozen in liquid nitrogen and stored at ⫺80°C. The K 2Q 40K 2 aggregate used for some experiments described in this paper was prepared in the same way as the Q28 aggregate. Preparation and storage of biotinyl-polyGln peptides. Biotinyl-K 2Q 28K 2 was solubilized and disaggregated as described above. After the determination of the exact concentration of the H 2O/TFA pH 3 stock solutions, the biotinyl-peptide was diluted into extension buffer to a final concentration of 10 nM, aliquoted, snap-frozen, and stored at ⫺80°C. Solutions of biotinyl-peptides stored at ⫺80°C develop over time small amounts of aggregated polyGln that give very high backgrounds in the extension assay. The stability of the biotinylK 2Q 28K 2 against storage-related aggregation was tested using the microplate assay (see below). In the absence of deposited aggregates in the wells, the signal given by biotinyl-peptide alone should not increase significantly after 5 h incubation under standard conditions. This signal background (corresponding to 0.2 fmol biotinyl-peptide) is unchanged until 1 month storage at ⫺80°C. After 1 month, however, backgrounds determined in this manner tend to increase. This increase can be very large, up to 4 –5 times higher than the normal background, if biotinyl-peptide is stored at high concentrations (500 nM). For this reason we prepare fresh stocks of biotinylated peptide every month, and store these stocks at relatively low concentrations (⬍60 nM) at ⫺80°C. The solubilization and preparation of biotinylK 2Q 40K 2 peptide were performed as described for biotinyl-K 2Q 28K 2. Preparation of aggregate plates. PolyGln aggregates were fixed to activated ELISA microtiter plates (EIA/RIA Plates, Costar, Atlanta, GA) by passive adsorption. The microplate was incubated uncovered for 17 h at 37°C with various amounts of K 2Q 28K 2 aggregate diluted in 100 ␮l extension buffer. During this time period the wells dry out. After 17 h incubation, the wells were washed 3 times with extension buffer, blocked for 1 h at 37°C with 0.3% gelatin in extension buffer, and washed again 3 times. Following this treatment, the plate was used immediately or could be stored for 1 week at 4°C with 200 ␮l of extension buffer in the wells. Most of the results presented in this paper were

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obtained with 20 ng of aggregates per well. The efficiency with which the above protocol supports the noncovalent retention of aggregates on the microplate wells was determined with the aid of the RP-HPLC standard. Thus, a 96-well plate was coated with 100 ng per well of K 2Q 28K 2 aggregates as described above. After 17 h incubation, and with no prior washing, 100 ␮l of extension buffer was added to each well and incubated 10 min. The 100-␮l supernatants (containing hypothetical nonabsorbed aggregate) were removed from each well, pooled, and centrifuged 30 min at 20,800g. This supernatant was discarded and the pellet was treated with 100% TFA 10 min, then diluted into water to give 0.1% TFA, and analyzed by RPHPLC. A standard curve was established in parallel with various known amounts of aggregates collected and treated in the same way. We determined that at least 96% of the aggregates were fixed to the microplate wells by this procedure. Extension assay. For each replicate of each time point, the extension/storage buffer was removed from an aggregate-coated well and replaced by 100 ␮l of 10 nM biotinyl-K 2Q 28K 2 peptide, and then the plate was incubated at 37°C. The kinetics data were collected by establishing individual time points in reverse temporal order. Thus, biotinyl-polyGln peptide was introduced into three wells (for reactions analyzed in triplicate) and the plate incubated at 37°C sealed with an adhesive overlay; these wells provide the longest time-point data. At this time, the original extension/storage buffer is retained for all other aggregate-coated wells. At the next appropriate time, the plate is removed from the oven and extension/storage buffer is removed and discarded from the next set of wells and replaced by fresh 100-␮l aliquots of 10 nM biotinyl-K 2Q 28K 2 peptide. The plate was resealed and returned to 37°C. This process was repeated until all time points were added. After a final incubation to provide the reaction time for the last-added (therefore earliest) time point, the entire plate was carried through the process described below to develop and measure the signal. Extension reactions were stopped simultaneously with 3 washes with extension buffer. After the 96-well plate was washed, it was incubated 1 h at room temperature with 100 ␮l per well of a 100 ng/ml europiumstreptavidin (EG&G Wallac, Gaithersburg, MD) solution in extension buffer containing 0.5% BSA. The plate was then washed 3 times with extension buffer and the europium was released from streptavidin by addition of 100 ␮l of enhancement solution (25) (EG&G Wallac). After 5 min, europium was measured by timeresolved fluorometry (26) in a Victor 2 (EG&G Wallac) 1420 Multilabel Counter using the programmed parameters for counting europium. Europium counts were converted to femtomoles europium using a stan-

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dard curve obtained with a europium solution obtained from EG&G Wallac. Femtomoles of europium was converted to femtomoles of biotinylated peptide using the manufacturer’s determination of 7 Eu 3⫹ ions per streptavidin molecule. It is possible that assay results might be compromised if biotinyl-peptides significantly dissociate from the aggregates during the europium-streptavidin incubation. However, we found in a control experiment that biotinyl-peptide deposited onto a polyGln aggregate coated well does not dissociate appreciably even after 5 h at 37°C (data not shown). Ataxin-3 (Q27) expression and purification. The human recombinant expression construct for the GST fusion with ataxin-3 (Q 27) [AT3(Q 27)], graciously provided by H. Paulson (U. of Iowa), contains an IPTGinducible tac promoter and a thrombin cleavage site providing for the efficient removal of the amino-terminal GST fusion from the AT3(Q 27) protein. The construct was transformed into the Escherichia coli expression strain BL21. The E. coli was grown at 37°C to late log phase (OD 600 ⫽ 0.8 – 0.9) before a 1 mM IPTG induction. Upon induction, the growth temperature was reduced to 32°C to minimize the deposition of the AT3(Q 27) into insoluble inclusion bodies. The culture was induced for 2 h before the cells were harvested by centrifugation. The pelleted cells were then resuspended in sonication lysis buffer (50 mM Tris, 50 mM NaCl, and 5 mM EDTA, pH 8) supplemented freshly with 0.15 mM PMSF, 1.46 ␮M pepstatin A, and 2.4 ␮M leupeptin (Sigma). The cells were subsequently lysed by sonication and the insoluble material was removed by centrifugation. Purification of the AT3(Q 27) protein was achieved through glutathione-Sepharose spin-filtration chromatography (Amersham Pharmacia, Piscataway, NJ) followed by on-resin cleavage with thrombin (Novagen, Madison, WI) to liberate the ataxin-3 from the bound GST fusion protein. As the final purification step, the resultant digest supernatant containing the cleaved ataxin-3 and thrombin proteins was applied to a S-300 Sephacryl (Amersham Pharmacia) gel-filtration column. The cleaved AT3(Q 27) eluted as a single peak at the void volume of the S-300 column, consistent with an average globular protein molecular weight of greater than or equal to 1500 kDa. This suggests the ataxin protein at this stage of the purification is a water-soluble aggregate containing at least 30 molecules of AT3. SDS-PAGE and Western blot screenings of the void volume peak revealed a single band at ⬃42 kDa that was immunoreactive with both an ataxin-3 polyclonal antibody (27) and the polyglutamine-specific 1C2 monoclonal antibody (Chemicon, Temecula, CA). These fractions were pooled and incubated at room tempera-

FIG. 1. Kinetic diagram of the extension of synthetic polyGln aggregate by biotinyl-peptide. A 96-well plate was coated with 20 ng/well of K 2Q 28K 2 aggregates and incubated with 10 nM biotinylK 2Q 28K 2. The y intercept of the linear fit of the slow phase represents the amplitude of the fast phase. Error bars reflect the standard deviation of 3 replicates.

ture for 6 days to complete the aggregation process. A sample of the aggregated AT3(Q 27) run on SDS-PAGE revealed a trio of SDS-resistant, Coomassie brilliant blue staining bands that were unable to be transferred and screened by Western blot (J. B. Hamilton, unpublished results). Subsequent dot blot screening verified that the aggregates retain immunoreactivity with the 1C2 antibody. The aggregates were pelleted by centrifugation and resuspended in 1X PBS, pH 7.4, buffer and stored at 4°C. Microtiter plates coated with aggregated AT3(Q 27) were prepared in analogy to the above protocol for chemically synthesized polyGln peptides. RESULTS

Figure 1 shows typical data obtained in the polyGln aggregate extension assay described above. The data are consistent with a two-phase kinetics model with a rapid first phase and a much slower second phase. Both phases are linear (r 2 ⫽ 0.97). Such kinetics define three parameters: the rate of the fast phase, the amplitude of the fast phase, and the rate of the second phase. We evaluated a slightly different set of parameters as possible endpoints for the assay: the rate of the first phase, the amplitude of the first phase, and the total amount of biotinyl-polyGln incorporated over a period of time sufficiently long (5 h) to include some second phase accumulation of label. Figure 2 shows that similar results are obtained whether the assay is used to follow the extension of aggregates of polyGln peptides either shorter or longer

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FIG. 2. Extension of polyGln aggregates with a benign or pathological length. Eighty nanograms per well of immobilized polyGln aggregates, K 2Q 28K 2 (A), or K 2Q 40K 2 (B) was incubated with 10 nM of either biotinyl-K 2Q 28K 2 (E) or biotinyl-K 2Q 40K 2 (F). The error bars represent the standard deviations obtained for 3 replicates.

than the pathological cutoff of 36 –38 Gln. Figure 2 also shows that similar results are obtained whether the biotinyl-polyGln peptide is shorter or longer than the cutoff. Further studies will be required to understand the basis for the quantitative differences in response with respect to peptide lengths, and to determine whether the differences observed are related to disease risk. However, the similar features for long and short peptides shown in Fig. 2 suggest that assays configured with either short or long peptides can probably be used, for example, to screen for inhibitors. Figure 3 shows that each of the three parameters exhibits an excellent linear correlation (r 2 ⫽ 0.99) with the weight of aggregate deposited on the microplate well, in the range from 5 to 80 ng of aggregate. This correlation is equally valid for extension of aggregates of polyGln peptides of lengths both below (Fig. 3A) or above (Fig. 3B) the pathological cutoff. Figure 4 shows that the rate of the fast phase also varies linearly with the concentration of biotinylated peptide in solution, over a range from 1 to 30 nM. Thus, the initial, fast phase of the extension reaction appears to be first order in both immobilized aggregate and tagged, solution-phase monomer. The assay behaves reasonably reproducibly on a dayto-day basis. Table 1 presents the values (averages of three replicates) of each of the three parameters obtained from three independent experiments done on different days. The modest day-to-day variation observed may be due to some decay in the stored plates over time, in variability between the plates themselves, or in some other factor. For best results in some applications, it may be important to conduct an experiment, with all appropriate controls, within the same time frame using the same materials. As elaborated under Discussion, it may also be possible to use this assay in a format that can detect and

FIG. 3. Linearity between the amount of the immobilized polyGln aggregate and the 3 different assay parameters. Various weights of the polyGln K 2Q 28K 2 (A) or K 2Q 40K 2 (B) aggregate were plated and incubated with 10 nM biotinyl-K2Q 28K 2 to determine kinetics of addition as shown in Fig. 1. Amplitude of the fast phase (■), rate of the fast phase (}), and total biotinyl-peptide bound (Œ). The errors bars are the standard deviations of 3 experiments conducted in triplicate.

FIG. 4. Dependence of the rate of the fast phase on biotinylpolyGln concentration. Twenty nanograms per well of immobilized K 2Q 28K 2 aggregates was incubated with biotinyl-K 2Q 28K 2, in the range from 1 to 30 nM. All of the kinetic data points were done in triplicate.

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Reproducibility of the Signal a

Rate of the fast phase (fmol/h) Amplitude of the fast phase (fmol) Total of biotinyl-K 2Q 28K 2 bound (fmol) a

Experiment 1

Experiment 2

Experiment 3

Average

1.8 0.8 1.3

1.5 1.2 1.7

2.0 1.1 1.6

1.76 ⫾ 0.3 1.03 ⫾ 0.2 1.5 ⫾ 0.2

A 96-well plate was coated with 20 ng/well of K 2Q 28K 2 aggregates and incubated with 10 nM biotinyl-K 2Q 28K 2.

quantify different levels of “extension-competent” aggregates. To explore this application, we coated a microplate with different amounts of K 2Q 28K 2 aggregate and incubated 4 h with a standard concentration of biotinyl-polyGln. Figure 5 shows that this assay exhibits excellent linearity of response (r 2 ⫽ 0.99), with a sensitivity sufficient for detection of as little as 80 pg of a synthetic aggregate. To confirm that the extension reaction observed for chemically synthesized polyGln peptides is guided by the same interactions as those operating in the aggregation of disease-related polyGln proteins, we prepared and tested aggregates of ataxin-3 (AT3), the protein responsible for Machado-Josef disease (28). We have found that chemically synthesized polyGln peptides shorter than the 35– 40 repeat length pathological cutoff are capable of forming ordered aggregates in vitro (S. Chen and R. Wetzel, unpublished). Similarly, AT3 with a normal length polyGln sequence has a strong tendency to form aggregates in vitro. As described under Materials and Methods, we purified AT3(Q 27) from E. coli extracts and allowed it to aggregate. We then investigated the ability of aggregates of AT3(Q 27) to be extended by biotinyl-K 2Q 28K 2 when the aggregates are fixed to microplate wells. In this experiment, the amount of AT3 aggregates fixed to the wells was adjusted so that the amount of polyGln on the

wells was the same as the amount of aggregated K 2Q 28K 2 on the same plate. Figure 6 shows that the kinetics of extension of both aggregates by biotinylK 2Q 28K 2 proceed with similar parameters. This suggests that the extension process we observe in vitro may be relevant to the disease process, and that we may be able to observe tissue-derived aggregates of disease-associated proteins using this assay. One important application of our microplate extension assay is for high-throughput screening of potential polyGln aggregation inhibitors. Compound libraries are often stored in DMSO, a powerful generic solvent. In addition, many chemical compound library entries will have acidic or basic functionalities and might thus alter the pH of the extension buffer in our assay. We therefore tested the assay’s sensitivity to DMSO and pH changes. The dependence of extension kinetics on DMSO concentrations was determined by plating K 2Q 28K 2 aggregates and incubating them with 9 nM of biotinylK 2Q 28K 2 peptide mixed with DMSO concentrations in the range of 0 –10%. Figure 7 shows that the three reaction parameters described above are not significantly affected by DMSO concentrations in the extension buffer up to 10%. Similarly, Fig. 8 shows that the

FIG. 5. Correlation of signal strength with the polyGln aggregate. Various amounts of K 2Q 28K 2 aggregates were deposited in the microplate in the 0.025–5 ng range, and incubated 4 h with 10 nM biotinyl-K 2Q 28K 2. The error bars represent the standard deviation of 3 replicates.

FIG. 6. Extension of a biological CAG repeat aggregate. Five nanograms per well of K 2Q 28K 2 (F) or 5 ng/well Q n -equivalent ataxin-3 Q 27 (■) aggregates was immobilized and incubated various times with 10 nM biotinyl-K 2Q 28K 2. The standard deviations of 3 replicates are represented.

MICROTITER ASSAY FOR POLYGLUTAMINE AGGREGATES

FIG. 7. Effect of DMSO concentrations on the deposition kinetics of the biotinyl-peptide. Wells containing 20 ng/well of plated K 2Q 28K 2 aggregates were incubated different times with 9 nM biotinylK 2Q 28K 2 mixed with 0 to 10% DMSO. Amplitude of the first phase (■), rate of the first phase (}), and total of biotinyl-K 2Q 28K 2 peptide bound (Œ). The error bars are the standard deviation of 3 replicates.

microplate assay can be used throughout the pH range 5.5 to 9 without dramatically affecting the extension reaction fast phase. DISCUSSION

Recent studies from a number of laboratories, including our own, have provided evidence for how polyGln length might independently contribute to disease risk, on the one hand, and to the mechanism of aggregate toxicity, on the other. In vitro fluorescence filtration assays with recombinant protein fragments (22) and fluorescence and light-scattering assays using chemically synthesized peptides (43) are consistent with data from animal and cellular models suggesting that there is a sharp increase in the ability of polyGln sequences to aggregate as repeat length increases from 35 to 40 Gln residues. At the same time, it is clear that polyGln sequences with repeat lengths in the 25–35 region are also capable of undergoing spontaneous, nucleationdependent aggregation, albeit with slower kinetics and less favorable thermodynamics compared to longer sequences (43). However, the fact that aggregation takes place more aggressively with repeat lengths above 35 suggests that the onset of significant disease risk at polyGln repeat lengths in the 35– 40 region may be related to the nucleation of polyGln aggregation in the cell. In contrast, the aggregation process that actually mediates polyGln toxicity may depend much less on repeat length effects. Recently, several groups have provided data from cell studies suggesting that polyGln aggregates are toxic in this family of diseases due to their abilities to recruit additional polyGln-

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containing proteins into the aggregate, and by doing so deprive the cellular environment of critical functions (21, and references cited therein). For example, recruitment of the CREB-binding protein, with its Q 19 sequence, into polyGln aggregates occurs efficiently and with toxic consequences in cells producing aggregates from a recombinantly expressed fragment of huntingtin (21). Supporting this result, we used the assay described here to show that polyGln peptides with repeat lengths of Q 15 to Q 20 can efficiently extend preexisting polyGln aggregates in vitro, while shorter peptides exhibit poor activity (43). Figure 2 shows that aggregates of either short or long polyGln peptides can support extension by either long or short biotinylpolyGln peptides. Together these results suggest that studies on the aggregation of both short and long polyGln peptides should prove valuable in Huntington’s disease research. First, because the aggregation process appears to be qualitatively similar for both short and long polyGln sequences. Second, because the recruitment of shorter polyGln sequences, once aggregation has been initiated by an expanded polyGln repeat protein, may in fact be a critical feature of the disease pathology. PolyGln aggregates formed in vitro exhibit features consistent with an ordered, amyloid-like assembly process (22, 23, 29). For example, polyGln aggregates appear to be highly ordered when evaluated by electron microscopy, and their formation in solution assembly reactions can be monitored both by light scattering and by the ability to confer amyloid-like (24) fluorescent properties onto the heteroaromatic dye thioflavin T (ThT). In fact, ThT binding and fluorescence can serve as the basis for a valuable polyGln aggregation assay (S. Chen, V. Berthelier, and R. Wetzel, manuscript in preparation). However, ThT assays have a number of

FIG. 8. Effect of pH on the rate of the fast phase. Wells containing 20 ng/well of immobilized K 2Q 28K 2 aggregates were incubated various times with 10 nM biotinyl-K 2Q 28K 2 at different pH values. The buffers used were 50 nM citrate at pH 4 to 6, 50 nM Tris-HCl at pH 7 to 8.5, and 50 nM glycine-NaOH buffer at 8.5 to 10. Each point represents the average of triplicate determinations.

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limitations for addressing both mechanistic questions and some practical applications. As discussed above, a major mechanistic concern with important consequences for polyGln toxicity mechanisms is the promiscuity of the polyGln aggregation reaction—the ability of other polyGln molecules in the environment to be recruited into a growing polyGln aggregate (43). Characterization of this recruitment process would be difficult with the ThT assay, since it responds equally well to all aggregated polyGln molecules. In addition, since it requires relatively high concentrations of peptide, use of the ThT assay as a screen for inhibitors of aggregation might be incapable of distinguishing between weak and strong inhibitors (30). Finally, solution-phase assays involving ThT and similar compounds suffer from the possible interference by test compounds with the assay readout. Based on these and other concerns, we developed the microtiter-based extension assay described in this paper. This assay is similar in format to an A␤ deposition assay described by Maggio and colleagues (31). This previously described assay utilizes 125I-labeled A␤ and, thus, is capable of detecting very low levels of addition of monomeric A␤ to a preexisting aggregate. In order to avoid using radioactivity, which raises safety issues and also introduces the inconvenience of requiring routine repeated syntheses of labeled peptide, we chose to use peptides tagged with biotin to follow the aggregation/deposition process. By using a streptavidin-europium complex and a time-resolved fluorescence microtiter plate reader to quantify the deposited biotin (26), we are able to observe very low, femtomole levels of polyGln deposition. The two-phase kinetic behavior we observed in the microplate assay (Fig. 1) is very similar to the that obtained for A␤ deposition by Maggio and collaborators, which they have interpreted in terms of a “dockand-lock mechanism” (31). According to this mechanism, the most recently added molecules of A␤ are relatively loosely bound and can still dissociate from the fibrils. A slow rearrangement, on the fibril surface, of this loosely bound A␤ to a more tightly bound conformation is required in order to create new binding sites for additional molecules of A␤. The similarity in reaction rate profiles between A␤ and polyGln deposition kinetics reinforces the idea that polyGln aggregates may have an amyloid-like aggregation pathway and substructure (22, 32). At the same time, the fact that A␤ fibrils do not support extension by biotinylpolyGln, and vice versa (V. Berthelier and R. Wetzel, unpublished), suggests that these aggregates share discriminating structural features that impart a significant degree of specificity onto the extension reaction. Especially in light of the dock and lock model for aggregate extension, we were concerned that we might lose significant amounts of bound peptide from the

aggregate during the 1-h incubation with europiumstreptavidin that is required by our detection system. However, control experiments showed that microplates with bound biotinyl-polyGln can be incubated for at least 5 h at 37°C with no detectible loss of signal. Similar experiments showed no loss of bound biotinyl-A␤ under similar conditions (R. Wetzel, unpublished). It is possible that some loosely bound biotinylpeptide is lost in the europium-streptavidin incubation, although the linearity of the first phase suggests that this is not significant. The europiumstreptavidin incubation is accomplished at 25°C, which may help to minimize dissociation during this step. The high degree of linearity between the amount of polyGln aggregate fixed to the plate and both the amplitude and the rate of the first kinetic phase (Fig. 2) is consistent with the dock and lock mechanism, which postulates a limited number of extension sites on the aggregate. Once all of these sites are occupied, further additions are not possible until the slow rearrangement process occurs that generates new sites (31). The rate of the first phase is also expected to be dependent on the number of extension sites available. In the same way, rates of A␤ fibril extension in suspension are dependent on the amounts of fibril seeds added to the reaction (33). The first order dependence of the fast phase rate on solution phase monomer concentration is also consistent with the dock-and-lock mechanism, and is inconsistent with any kind of concentration-dependent preequilibrium to form a required, extension-competent multimeric intermediate. The two-phase kinetics, and the linear proportionality between the immobilized polyGln aggregates and the three kinetic parameters, were also seen for aggregates containing a longer repeat of 40 glutamines. Variations of this microplate assay should have many applications. The basic assay will allow studies on the effect of various solution parameters, such as ionic strength, metal ions, temperature, and pH, on reaction parameters. Along with polyglutamine sequence length, other aspects of polyGln-containing proteins may influence the tendency of these molecules to aggregate. The microplate assay can be used to study structure-function relationships of the polyGln aggregation reaction in a clean, well-defined system. For example, we have been able to show that the recruitment of biotinyl-polyGln peptides into a preexisting polyGln aggregate is dramatically length dependent (43). This biophysical data may facilitate our understanding of the molecular mechanisms of polyGln cytotoxicity. The assay described here depends on the ordered, amyloid-like assembly of polyGln peptides, mediated by noncovalent forces, into ordered aggregates. There is as yet no consensus on the extent to which brain aggregates of polyGln exhibit amyloid-like substruc-

MICROTITER ASSAY FOR POLYGLUTAMINE AGGREGATES

tures (32, 34). It is also not known to what extent aggregation is mediated by modifications by transglutaminase (35–37), interaction with chaperones (38, 39), etc. Further characterization of brain-derived aggregates, including work with derivatives of the assay described here, should help to elucidate these points. Should in vivo aggregation prove to play an important role in the pathology of polyGln diseases, one possible means of attacking the disease mechanism therapeutically is by developing anti-aggregation compounds. The features of the microplate assay described here suggest it may be a valuable tool in screening compound libraries for such inhibitors. One particular strength of the assay is the small amounts of material required for each determination. This not only ensures that the assay will be economical, but it also suggests that the assay will have the dynamic range capable of distinguishing between weak and strong inhibitors. [That is, when an assay requires a relatively high concentration of the molecular target, even strong inhibitors only exhibit significant activity when they are assayed at concentrations comparable to that of the target; this titration effect masks the true potency of the inhibitor (30)]. The validity of the microplate assay for addressing issues on expanded CAG repeat diseases is supported by its ability to detect biologically relevant AT3(Q 27) aggregates. Although the aggregates used here are from a benign-length polyGln sequence, it is known that such sequences can give rise to aggregation and formation of intranuclear inclusions in vitro when they are targeted to the nucleus (40, 41). The specificity of the assay for polyGln peptide extension and its high sensitivity should allow its use for detection of extension-competent microaggregates in tissue extracts, cell fractions, and biological fluids. This in turn will make possible mechanistic studies to determine the relationship of polyGln aggregation to CAG repeat pathogenesis, and may also provide the basis for a clinical diagnostic test for disease onset and progression. Protein aggregation is increasingly suspected to be an important causative factor in human diseases, including a number of neurodegenerative diseases (42). Historically, protein aggregation and precipitation reactions have been viewed as nonspecific processes that are intrinsically unapproachable by analytical and biophysical methodologies. However, recent focus on the protein biophysics of amyloid diseases such as Alzheimer’s disease has revealed that at least some diseaserelated protein misassembly processes are guided by regular structural interactions that bestow a certain specificity to the assembly process. The assay format reported here for polyGln aggregate extension may have general applicability for the study of the basic molecular features, as well as the inhibition, of other protein aggregation reactions.

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ACKNOWLEDGMENTS This work was supported by a Lieberman Award and a Cure Huntington’s Disease Initiative grant from the Hereditary Disease Foundation, and by the Lindsay Young Alzheimer’s Disease Research Gift Fund. We thank Dr. Henry Paulson for the gift of AT3 expression clones. We also gratefully acknowledge Stephen Wood for suggesting the detection method used in this assay.

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