Amyloid precipitation in biofluids using a structure-specific chemical antibody

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. We introduce the structure-specific chemical antibody; a Y shaped, bioinspired small molecule with a dimeric region to mimic avidity, and an attachment region to mimic the Fc region. Our probe, capture molecule for amyloid precipitation (CAP-1), consists of a derivative of Pittsburgh compound B (dimer) to target the cross  -sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we targeted the amyloid structure of protein aggregates in human cerebrospinal fluid, isolated them for analysis and then characterised them using single-molecule fluorescence imaging and mass spectrometry. AP allows unbiased determination of the molecular composition and structural features of the in vivo aggregates, formed in neurodegenerative diseases, that are present in biofluids.


Introduction
-Synuclein, amyloid- and tau are examples of proteins that self-aggregate in cross -sheets motifs, and are present in Lewy bodies, amyloid plaques, and tau tangles, respectively 1 . These cross -sheets (or amyloid structures) are found in the brains of people with neurodegenerative diseases such as Parkinson's and Alzheimer's disease [2][3][4] . Importantly, brain extracts containing misfolded amyloid- from patients with Alzheimer's disease (AD) and preformed -synuclein fibrils induced cerebral β-amyloidosis and -synuclein propagation, respectively and associated pathologies in mice 5-8. On the other hand, depletion of aggregates from an AD brain suppressed in vivo seeding capability 9 reinforcing the idea that the induction of pathology is likely governed by the structure and concentration of the aggregate seeds 10,6 and highlights the importance of studying the aggregated protein as opposed to its monomeric counterpart.
The exact mechanism by which protein aggregates lead to progressive loss of neuronal cells and result in subsequent pathophysiologic effects like dementia and movement disorders remains poorly understood. It is known that subtle differences in amino acid content result in major structural changes that have an impact in the pathophysiology of these diseases [11][12][13][14] . Also, in vitro studies have revealed that protein aggregation is a dynamic process where a wide range of aggregates with variable sizes 1 and hydrophilicities 15 are formed, and that the aggregates become more toxic when they acquire cross -sheets structure 1,16 . Small soluble protein aggregates (< 200 nm) of synuclein and amyloid- as well as, late insoluble tau aggregates are implicated in cellular cytotoxity [17][18][19][20][21][22][23] . Moreover, it was recently reported that small soluble amyloid- aggregates induced extensive membrane permeability while larger -sheet containing aggregates were most effective at causing an inflammatory response in microglia cells 22 . These findings were replicated in a recent study of the aggregates present in cerebrospinal fluid (CSF) of patients at different stages of AD 24 .
The aggregates in the CSF of mild cognitively impaired patients induced. more membrane permeabilisation, while larger -sheet aggregates present in the CSF of AD patients were more effective at inducing inflammation 24 . Together these studies reinforce the idea that aggregates of Finally, we determined the binding affinity of CAP-1 to -synuclein and compared this affinity with that of ThT using bulk fluorescence and mature sonicated fibrils (average length 200 nm), to avoid heterogeneity in the structure and size of the aggregates, see SI.14. Using an initial concentration of 100 nM -synuclein we obtained a Kd (CAP-1) = 82.63 ± 11.70 nM and Kd (ThT) = 3962 ± 352 nM ( Fig.1e), representing a 50-fold increase in affinity of CAP-1 compared to ThT. Dissociation constants often depend on the approach used and previous studies reported Kd of ThT for synuclein fibrils from 588 nM to 100 M 39,40 . In a study using a similar methodology to ours but using a40 fibrils instead of -synuclein the Kd was 2.3 M 37 . The significant increase in CAP-1 affinity towards amyloids compared to ThT can be explained by the combination of two key factors: CAP-1 being a dimer, as previously described avidity increases affinity 37 and the absence of the Nmethylated benzothiazole moiety (SI.1) as seen for PiB 35,36 . (b) Illustrative diagram highlighting the selective affinity of CAP-1 to cross β-sheets present in early stage aggregates and fibrils but not in monomers. (c) TIRFM images of α-synuclein aggregation at 0h, 8h (red circles highlighting oligomers) and 24h using 5 µM CAP-1, ex405 nm. Scale bar = 5 m, inset scale bar = 2 m. (d) Fluorescence intensity increase of CAP-1 (20 µM) upon binding to 10 µM α-synuclein at different time points of the aggregation reaction using ex355 nm. The error bar represents the SD of the maximum fluorescence intensity between two independent experiments. The p-value corresponds to the result of a one-way ANOVA, and Tukey's post hoc comparison. **p<0.0017, ***p=0.0007, n.s. p>0.05. (e) Binding affinity of CAP-1 and ThT to α-synuclein. Increasing amounts of CAP-1 or ThT were added to 100 nM α-synuclein. The Kd was obtained by fitting the experimental points to a hyperbolic curve (specific binding), Kd (CAP-1) = 82.63 ± 11.70 and Kd (ThT) = 3962 ± 352 nM.

Capture of protein aggregates using CAP-1 -Method of amyloid-precipitation
Following the characterization of CAP-1 binding to -synuclein, we designed a protocol for isolation of protein aggregates from solution, which we have named amyloid precipitation (AP).
The schematic of AP is outlined in Fig. 2a-b. After the conjugation of CAP-1 with magnetic streptavidin-coated beads (Fig. 2a), the beads are added to a solution containing protein aggregates such as recombinant -synuclein solution or a biofluid. After 2h at 4C with gentle mixing, the beads are separated using a magnet and both fractions ('beads' and 'depleted') are analysed by TIRFM (Fig. 2c) and bulk fluorescence (Fig. 2d). The presence of fibrils (right panel) attached to the beads is visible by the 'hairy' appearance of the beads and highlighted in the magnified bead, and contrasts with the plain look of beads without protein (left panel). Despite the heterogeneous bead-to-fibril attachment, some beads contain many small fibrils and other fewer but longer fibrils , there is a significant difference in the diameter (measured as fluorescence intensity profile) between beads in the presence or absence of −synuclein fibrils, 105 nm (p = 0.0002) confirming the successful binding of fibrils to beads (see SI.15).
In Fig. 2d we tested the efficacy of AP towards -synuclein fibrils (purple) versus -synuclein monomers (orange) (see SI.16 for representative fluorescent spectra of both fractions). The low fluorescence intensity detected for the supernatant of both samples, 11% for fibrils and 3% for monomers, respectively, reflects the presence of residual CAP-1 molecules released from the beads during the incubation and as expected is higher for the sample containing fibrils. The difference between beads incubated with -synuclein fibrils (100%, purple) and beads with -synuclein monomers (33%, orange) highlights the absence of cross -sheet in the monomeric solution and corresponds to the fluorescence of CAP-1 alone. The fluorescence intensity for the samples, beads+CAP-1+monomers (Fig. 2d orange) and beads+CAP-1+PBS (blank, see SI.16), is the same for both beads and supernatant, confirming that CAP-1 does not bind to monomers (see SI. 16).
Overall, the fluorescence increase between the supernatant (11%) and beads (100%) for the synuclein fibrils sample demonstrates the successful pulldown (and concentration) of aggregates by the beads.
In both TIRFM (Fig. 2c) and bulk (Fig. 2c) measurements, detection of protein aggregates is based on CAP-1 intrinsic fluorescence, highlighting its ability to strongly bind (capture) aggregates and work as optical readout for the presence of -sheets. We also used atomic force microscopy (AFM), an orthogonal non-optical technique, to confirm the successful binding of -synuclein fibrils to CAP-1-beads (SI.17). As shown in the 3D (height) image fibrils localize preferentially close to the beads (SI.17a), once more demonstrating the preference of protein aggregates to CAP-1 coated beads.
Until now, we have used mature -synuclein fibrils (sonicated 200 nm, non-sonicated >1 m) as a model of protein aggregation to test AP. However, in biological fluids such as cerebrospinal fluid (CSF) the amyloids present are smaller. These 'early stage' aggregates, or oligomers, have been shown to be much smaller that the optical diffraction limit (~250nm) 38 and confirmed to be approximately tens of nanometres in size using higher resolution methods such as AD-PAINT 15 and AFM 41 . For this reason we used -synuclein aggregates collected at the 8 hour time point to maximize the number of oligomers 15,16 and to validate the AP method for use in a biological context.
In the presence of CAP-1 the number of protein aggregates in solution after pulldown is reduced to background levels (Fig. 2e, grey column -4.2 ×10 -4 ± 2.3 ×10 -4 /m 2 ). In the absence of CAP-1 there was partial removal of aggregates (see SI. 19) suggesting unspecific binding to the beads but negligible compared to the virtually complete depletion, 99.4%, in the presence of CAP-1.
Next we investigated the use of mass spectrometry (MS) to quantify the amount of -synuclein enriched on the beads after pulldown as MS will allow identification of molecular composition of the amyloids captured using AP. For this, we used high-resolution parallel reaction monitoring (PRM) mass spectrometry (MS). After AP, -synuclein was eluted from the beads and digested using trypsin, converting full length -synuclein into small peptides, namely -syn13-21, -syn35-43, -syn46-58, -syn61-80, and -syn81-96, see Fig. 2. The PRM-MS spectrum in Fig. 2g shows the relative abundance of each peptide. In order to confirm the specificity of CAP-1, we compared the presence and absence of CAP-1 during the AP. In the presence of CAP-1 the amount of individual peptides was 5 to 13 times higher, depending on the peptide, than without CAP-1 ( Fig. SI.21). This is in agreement with TIRFM results (Fig. 2e). The error bar represents the SD of the maximum fluorescence intensity between two independent experiments (each made in triplicate) and differences between two groups were analysed using unpaired two-tailed Student's t test, *p=0.0345, **p=0.0062 and ***p=0.0004. (e) Depletion of -synuclein oligomers (time point 8h of -synuclein aggregation reaction) by AP and quantification of aggregates left in the supernatant (depleted fraction). Plotted is the fluorescent puncta counts x10 2 /m 2 for the sample before and after AP using TIRFM. AP captures approximately ~100% of oligomers in solution. The error bar represents the SD of the number of fluorescence puncta between at least 27 fields of view for one representative experiment (see SI.19 for TIRFM images) (f) Outline of AP followed by on bead digestion. -synuclein13-21 peptide fragment ion PRM spectrum in the presence (right) and absence (left) of CAP-1, recovered after AP from a solution containing 1 nM total -synuclein (<50 pM oligomers).

Amyloid-precipitation followed by PRM mass spectrometry of -synuclein spiked in human CSF
AP is an unbiased method to capture amyloid protein from solution, allowing subsequent mass spectrometry identification of proteins present in such aggregates 42,43 . As CSF is a complex biofluid made of more than two thousand different proteins 44 , we firstly determined the sensitivity of CAP-1-beads to capture known amounts of -synuclein spiked in CSF.
Increasing amounts of either purified -synuclein monomers (t = 0 hours) or -synuclein mixture of monomers (>95%) and oligomers (<5%) 16 (t = 8 hours), were spiked in control CSF, see Fig. 3a for the outline of the experiment and SI.22 for TIRFM representative images. After AP, the beads were trypsin-digested and analysed by PRM-MS. In Fig. 3b the amount of -synuclein13-21 peptide recovered as a function of the initial -synuclein concentration spiked is shown. Naturally occurring -synuclein oligomers present in CSF were undetectable (see Table 3-8 for list of proteins pulled down). For concentrations equal to and below 1 nM, monomers were not detected, while in 1 nM of mixed species 28 femtomoles (28 pM) of -syn13-21 captured were detected (SI.21 for other peptides). For -synuclein concentrations higher than 1 nM, the increase in -syn13-21 detected is linear and about three times higher for the mixed species sample than for the monomers (Fig. 3b).
CAP-1-beads captured 0.6% of total -synuclein monomers (orange) spiked in CSF and 2.3% of total -synuclein mixture (monomers >95% and oligomers <5%, purple). This means that almost no monomers in solution are captured while approximately 50% (2.3% out of <5%) of the oligomers added to CSF are captured. For this reason, the 3-fold change in the total amount of -syn13-21 recovered (Fig. 3b) represents a large difference in capture affinity between the monomer which is present at high concentration and low concentration of aggregated -synuclein. This result confirms the specificity of AP in capturing protein aggregates compared to the monomers in complex biofluids such as CSF.
The proteins captured using CAP-1 beads should be enriched in amyloid prone proteins or contain proteins in the CSF that bind amyloid proteins 32 . Using PASTA 2.0 45 and RFAmyloid 46 , two highly cited web servers for the prediction of protein aggregation from sequence we observed an increase in the total number of amyloid-prone proteins when using CAP-1 compared to unmodified beads (see SI Table 9). As expected, in the presence of CAP-1 there is an increase in the total -strand content of captured proteins (25-26%) compared to not using the capture molecule (19%), providing further computational evidence of the ability of CAP-1 to select -sheet containing proteins (see Table 1 and 2).
To evaluate the efficiency of AP in removing toxic amyloid species from CSF we used a sensitive membrane permeability assay previously developed 18 (see Fig. 3c for outline of the experiment).
CSF is diluted in a solution containing Ca 2+ and then added to liposomes containing a Ca 2+dependent dye encapsulated. If CSF contains amyloids/oligomers that cause membrane permeability, Ca 2+ enters the liposome resulting in increased fluorescence. In Fig. 3d, the average Ca 2+ influxes for CSF before AP (purple), CSF after AP ('depleted' fraction) (white) and CSF after AP without CAP-1 (white with purple dots) using the same CSF as in Fig. 3b it is plotted. AP removed most of the CSF proteins responsible for Ca 2+ influx, reducing membrane permeability from 27% to 6%. Having established that AP is able to remove amyloid proteins from control CSF ( Fig. 3b-d), we then decided to use CSF from Parkinson's disease (PD) patients in a separate set of experiments ( Fig. 3e-f). TIRFM images showed a significant decrease in the number of ThT active species after AP Fig. 3g (left panels) and we found that there was a reduction of ~50% in Ca 2+ influx.
This demonstrates that AP can capture amyloid aggregates from PD CSF. It is worth noting that there is non-specific binding to the beads without CAP-1 that leads to some aggregate capture and a small reduction in membrane permeability (Fig. 3d-e middle column).

Conclusions
Protein aggregates have been known to be implicated in neurodegenerative diseases for more than three decades 47  In this study, we presented the synthesis and characterisation of a structure specific chemical antibody designed to capture the protein aggregates associated with neurodegeneration from solution. This molecule has been specifically developed to bind and isolate a target molecule based on secondary structure, the presence of -sheets, using chemical head groups that form the basis of PET ligands 53,54 . Previous studies have made use of dimerised ligands (protein/peptide 55,56 or ThT 37 ) as a way to improve binding affinity to a particular target molecule. To the best of our knowledge, this is the first study to exploit the increased affinity of dimerised ligands in order to enable isolation/precipitation of the target species based on its structure rather than its protein composition.
The CAP-1 structure was successfully designed and then demonstrated to bind and isolate aggregates with amyloid structure (oligomers and fibrils of a  , tau and α-synuclein), but crucially not monomers, using synthetic aggregates. However, it is also critical to demonstrate translational relevance. In vivo, the complexity of the biofluids that surround the CNS (CSF) and the brain tissue itself makes the detection of small amyloids a major challenge 57 . We demonstrated the sensitive detection of amyloid containing aggregates can be performed in CSF by amyloid pulldown using CAP-1, followed by bead digestion and detection by mass spectrometry. We observed an increased number of amyloid-prone proteins when using CAP-1 compared to plain beads and an increase in the total content of -strand highlighting the strength of our method AP in enriching -sheet containing proteins (see Table 1 and 2).
This new capability for unbiased detection and capture of amyloids, coupled with a mass spectrometry approach to provide the molecular composition is a powerful combination because it has the potential to define the amyloids present in the brain, CSF and other biofluids.
The ability of disease causing aggregates (containing -sheets) to cause membrane permeabilization as been previously correlated with their cytotoxic potential 18,22 . Herein, we demonstrated that we could capture the aggregates responsible for membrane permeabilisation present in CSF, reinforcing the idea that AP is an efficient tool to deplete toxic aggregates from biofluids. Importantly, in AP the beads contain negligible monomers and, since it is possible to remove the captured aggregates from the beads, this approach allows further characterization of the human derived aggregates and cytotoxicity experiments to be performed.
Further improvements to the design of the capture molecule are possible by optimising the linker length, head groups and synthesising multimeric molecules to further improve the sensitivity and selectivity of amyloid precipitation 58,59 . There may also be significant advantages in this approach in terms of stability and resistance to degradation compared to conventional antibodies 60-62 .
In conclusion, we have successfully developed a new molecule inspired by the trimeric shape of an antibody to target all aggregates containing cross-beta sheet motifs present in complex biofluids.
CAP-1 has two binding sites to improve avidity and a third moiety to enable surface immobilisation and therefore capture of aggregates based on their structure, but not their protein composition. This simple and versatile method allows the identification of molecular components of aggregates, using mass spectrometry. Overall, this structure-based approach will pave the way to understanding the exact molecular species responsible for neurodegeneration in humans and consequently hasten development of simple and robust early diagnosis methods.

Aggregation of α-synuclein
Monomeric wild-type α-synuclein was purified from Escherichia coli as previously described 63 .

Amyloid precipitation assay -AP
The amyloid precipitation (AP) assay consists of the pulldown of protein aggregates (e.g. synuclein) using streptavidin-Dynabeads (MyOne TM Streptavidin C1, Invitrogen) conjugated with CAP-1. Briefly, 30 L of beads/sample were removed from the vial, resuspended in 1 mL PBS and placed on a magnet for 2-3 min for separation and the supernatant discarded (this step was repeated three times). Afterwards, the beads were resuspended in 1 mL of CAP-1 30 μM and the tube placed in a revolver mixer for incubation at room temperature during 1h. Following, the tube was placed on the magnet for 2-3 min and the supernatant discarded. The beads were washed three times with PBS as before. Finally, the beads were resuspended in 500 μL solution containing -synuclein 10 μM (monomers alone, or mixture of aggregates) and left in the revolver mix for 2h or overnight at 4 C. In the end, the tube was placed on the magnet for 2-3 min and 450 μL of supernatant were removed to a clean tube and labelled as 'depleted' fraction, both the depleted fraction and the 'beads' PBS buffer (Whatman Anatop 25 0.02 μm). Each batch of coverslips was tested for fluorescent artefacts (i.e. false positives) by imaging thioflavin T (ThT) 5 μM. ThT stock solution was prepared as described elsewhere 38 and ThT working solution (50-100 μM) was filtered (Whatman Anatop 25 0.02 μm) prior to use and concentration determined using  412 nm 36000 M -1 cm -1 .

Total internal reflection fluorescence microscopy (TIRFM) imaging
Imaging was performed using a homebuilt total internal reflection fluorescence microscope as reported previously 38

AP of -synuclein spiked in CSF followed by on-bead digestion
The CSF sample aliquots used were de-identified leftover aliquots from clinical routine analyses, following a procedure approved by the Ethics Committee at University of Gothenburg (EPN 140811 (ThermoFisher Scientific) and Pinpoint 1.3.0 (ThermoFisher Scientific) for determining selected fragment ion peak areas, respectively. The MS accuracy was ± 10 ppm centred at 0, a MS/MS accuracy of ± 15 ppm and the isolation mode set to MS/MS with an isolation width of 3.0 u. The peaks were detected using a chromatographic peak with a window size of ± 2.0 min. The complete peak area was determined after using four points of smoothing. The scheduling window size for identified transitions was ± 0.5 min. The detected fragment ion peaks were manually inspected for accuracy and absence of interferences from other peptides than the peptide of interest, including fragments originating from other product ions in the same pair/triplet. The relative amount of spiked unlabelled or 15N-labeled α-synuclein peptide was calculated by normalizing the measured peak area with the peak area of the corresponding IS peptide. where P is the probability that the observed match is a random event.

Membrane permeability assay
Details of this method have been described previously 18 . Studies have shown that single vesicle assay can be used to measure the toxicity of β-sheet rich protein aggregates present in CSF 66  into an individual vesicle due to protein aggregates present in CSF was then determined as The average degree of was calculated by averaging the Ca 2+ influx into individual vesicles. The membrane permeabilization experiments were performed using a homebuilt TIRF imaging setup microscope using 1.49 100× Nikon TIRF Objective. For excitation 488-nm laser (Toptica) beam and images were acquired using an air-cooled EMCCD camera (Evolve Delta).

Data analysis
Microscopy images were analysed using ImageJ and Matlab. GraphPad Prism 8 was used for statistical analysis, plotting and curve fitting. Statistical analysis was performed using unpaired twotailed Student's t test to analyse differences between two groups, or a one-way ANOVA and Tukey's post hoc comparison to analyse differences among three or more groups. Differences were considered to be statistically significant if p < 0.05. To determine the number of fluorescent puncta in each image an average of the entire stack was generated and used to detect each protein aggregate using the Find Maxima function in ImageJ (with a threshold value of 180 Figure 2e). To compare proteins pulled down from control (A) and PD (B) CSF in the presence (1) and absence (2) of CAP-present in all three replicates and with at least 2 unique peptides. Then we compared list A1 with A2 and B1 with B2. See SI Table 3 -8 for results: List of common proteins (Table 3 (A1-A2) and   Table 6 (B1-B2)) and list of exclusive proteins (  (Table 9), two bioinformatic tools to predict % of -helix and % of -strand, and amyloid formation, from protein sequence analysis, respectively.