ABSTRACT Protein aggregation is a hallmark of several degenerative

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Selective and sensitive pull down of amyloid fibrils produced in vitro and in vivo by the use of pentameric-thiophene-coupled resins. Anna Beatriz Wreden, Luiza Fernandes, Mirian Kelley, Antônio PereiraNeves, Caroline S. Moreira, David R. da Rocha, and Fernando L Palhano ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00222 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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ACS Chemical Neuroscience

ABSTRACT Protein aggregation is a hallmark of several degenerative diseases, including Alzheimer’s disease, Parkinson’s disease and familial amyloidosis (Finnish type) (FAF). A method to isolate and detect amyloids is desired for the diagnosis of amyloid diseases. Here, we report the synthesis of pentameric thiophene amyloid ligand (p-FTAA) linked to agarose resin for selective purification of amyloid aggregates produced in vitro and in vivo. Using amyloid fibrils produced in vitro from alpha-synuclein, gelsolin and Aβ1-40 and gelsolin amyloid aggregates extracted from tissue homogenates of a mouse model of FAF, we observed that p-FTAA resin was able to pull down amyloid aggregates. The functionalized resin was also able to pull down oligomers produced in vitro from the A30P variant of alpha-synuclein. The methodology described here can be useful for the diagnostic of amyloidogenic disease and also can be used to purify amyloid fibrils from biological samples, rendering the fibrils available for more accurate structural and biochemical characterization.

INTRODUCTION Protein aggregation is implicated in more than 40 human diseases1. A common feature of these diseases is the presence of amyloid aggregates. Amyloid diseases are named after the amyloid fibril deposits exhibiting a cross-β-sheet structure that are found in patients afflicted with these maladies2. The high stability of the amyloid quaternary structure renders amyloids difficult to degrade; consequentially, amyloid isolation from natural materials is tremendously challenging3. Amyloid aggregates are also found in several organisms, such as bacteria and fungi, where they perform physiological functions; these are known as functional amyloids4-6. The diagnoses of amyloidogenic

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diseases are based on clinical evaluation by physicians and by biopsies of suspected tissues stained with the amyloidogenic probe Congo red2. When bound to amyloid fibrils in vitro and in situ, Congo red exhibits several spectroscopic features that can be used for diagnostic purposes, such as apple-green birefringence under polarized microscopy, red fluorescence under fluorescence microscopy, and a blueshift in the absorbance spectra2. Clinicians generally use the apple-green birefringence as a gold standard method for amyloid detection7. However, Congo red staining of biopsies is a method with low specificity and sensitivity since other molecules, such as collagen, can be identified as false-positive amyloids, and the detection is dependent on the operator of the microscope8. Hence, a specific and sensitive protocol to isolate and detect amyloids is much needed for the diagnoses of amyloid diseases9. Additionally, a protocol to isolate amyloid fibrils would be useful for the discovery of new functional amyloids7. Several different strategies, such as immunoprecipitation3, fluorescence correlation spectroscopy10, peptoid capture reagent assays11, and affinity capure12 have been used to isolate and detect amyloid fibrils from complex solutions, but none of them have been broadly applied or had high specificity and sensitivity. Luminescent conjugated polythiophenes have been used to study protein aggregates in vitro, in situ and in vivo1315

. The major advantages of thiophene-based probes over conventional amyloid ligands, such as

Congo red, thioflavin T and derivatives, are their ability to identify a broader subset of diseaseassociated protein aggregates, their higher sensitivities and their utility in spectroscopic assignment of heterogeneous populations of deposits or distinct protein aggregates16. Our aim was the development of a resin where small-molecule compounds with high affinity for amyloids could be covalently coupled to pull down fibrils from complex solutions. For this purpose, one thiophene derivative, pentathiophene (p-FTAA) (Fig. S1), presents another advantage when compared with Congo red and thioflavin T. As shown in Figure S1, both Congo red and thioflavin T present few

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reactive groups in their molecules. On the other hand, p-FTAA presents four carboxyl groups per molecule with a theoretical pKa ranging from 3.46-4.35 and is potentially reactive. Here, we synthesized an agarose resin covalently bound to p-FTAA in order to purify amyloid fibrils. For this purpose, we used three different sources of amyloid fibrils produced in vitro, namely, α-synuclein, gelsolin and Aβ1-40, as well one source of amyloid fibrils produced in vivo, in this case, the muscle lysate of the mice that overexpress the human gelsolin protein. Our data show that our protocol was able to purify microgram quantities of amyloid fibrils (produced in vitro and in vivo) present in complex solutions.

RESULTS AND DISCUSSION We used a cross-linked beaded agarose support containing reactive primary amines at the end of a long spacer arm to couple the p-FTAA through its carboxyl groups (Fig. 1A and Fig. S2). We incubated p-FTAA at pH 5.0 with [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl] (EDC). EDC reacted with the carboxylates from p-FTAA to form an acylurea active ester that was reactive with nucleophiles such as the primary amines on the agarose resin (Fig. S2). The final product was a resin coupled covalently with p-FTAA, denominated p-FTAA-R (Fig. 1A). In the subsequent experiments, we used as a negative control an agarose resin where no coupling had been performed (nc-R). It is important to note that the coupling is not selective and can occur with any of the fourcarboxyl groups of p-FTAA (Fig. 1A). Moreover, a single p-FTAA could possibly be coupled with one or more amine groups of the resin, cross-linking the resin (Fig. 1A). To very this possibility, we analyzed the morphology of the beads by light-microscopy looking for dimeric, trimeric or other complex aggregates. As observed in Figure S3, more than 85% of the p-PFTAA-R beads were loose, that is, not cross-linked with other beads. We used as a negative control the nc-R, and the

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same result was observed (Figure S3). We conclude that the coupling of p-FTAA in to agarose resin was not able to cross-link the resin. Using scanning electron microscopy, we observed no surface alterations to p-FTAA-R when compared with nc-R (compare Fig. 1F with 1G). The p-FTAA-R showed green fluorescence (Fig. 1B and Fig. 1H), while no fluorescence was detected in the nc-R (Fig. 1D and Fig. 1H), confirming the coupling of p-FTAA to the agarose resin. When free in solution, p-FTAA molecules possess low green fluorescence that is substantially increased by the presence of amyloid aggregates (13 and Fig. 2A). We hypothesized that p-FTAA-R could be used to pull down amyloid fibrils from complex solutions, and their fluorescence could be used as an indicator of the presence of amyloids. We tested if the presence of amyloid fibrils of alphasynuclein (which is described in detail later in the manuscript and in Figure 2) could enhance the green fluorescence of p-FTAA-R, but we observed no change (Fig. S4A). One possible explanation for this observation is that the immobilized p-FTAA molecule probably experiences a hydrophobic environment when attached to the agarose resin, leading to a high fluorescence quantum yield. Hence, the binding of any amyloid fibrils would not be sufficient to promote an increase in its fluorescence. However, when nc-R was added in a solution containing free p-FTAA we observed no change in fluorescence, suggesting that just the hydrophobic environment, or the interaction of the negatively charged p-FTAA with the cationic resin are not enough to change p-FTAA properties (Fig. S4B). Even though the interactions of anionic groups of oligothiophene have been implicated in the interaction with amyloid and the specific spectral properties of the molecule172, another explanation for the differences in the fluorescence behavior of immobilized p-FTAA may reside on the changes in molecule dynamic caused by resin ligation. Other research groups have made substantial progress in the knowledge of fluorescent properties of thioflavin T (Fig. S1B). Upon binding to the surface of amyloid fibrils, the twisting motion of the central C-C bond that connects

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the two rings (Fig. S1B) is hindered, enhancing the thioflavin-T fluorescence18. Therefore, changes in the fluorescence properties of p-FTAA-R could be caused by the immobilization of p-FTAA, restricting the motion of the molecule in a similar way that happening with thioflavin-T. We concluded that p-FTAA-R cannot be used to detect amyloid fibrils due to enhancement in its fluorescence quantum yield (Fig. S4A) as compared to that of free p-FTAA (13 and Fig. 2A).

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Figure 1. Characterization of p-FTAA and non-coupled resins. (A) The p-FTAA molecule was coupled to agarose resin by a covalent bound between a carboxyl group and a primary amine present in the long arm of the resin, resulting in an amide bond. The coupling can occur in any of four carboxyl groups of p-FTAA. The p-FTAA-coupled resin (p-FTAA-R) (B and C) and non-coupled resin (nc-R) (D and E) were visualized by fluorescence microscopy in a green channel (B and D) or by phase contrast (C and E). Scanning electron microscopy of nc-R (F) and p-FTAA-R (G). Inset: magnification of panel G. (H) Both resins (nc-R and p-FTAA-R) were excited at 450 nm in a fluorimeter, and the emission spectra were collected between 480 and 700 nm.

To test if p-FTAA-R could capture amyloid fibrils, we produced amyloid fibrils using three different proteins, namely, alpha-synuclein, an 8 kDa fragment of gelsolin, and Aβ1–40. Alpha-synuclein is associated with Parkinson’s disease19, gelsolin with familial amyloidosis (Finnish type) (FAF)20, and Aβ1–40 with Alzheimer’s disease21 (Fig. 2). The fibrils formed by the three different proteins presented the typical amyloid structure (Fig. 2C, 2D and 2E), as seen by transmission electron microscopy (TEM). All fibrils were further characterized by p-FTAA fluorescence (Fig. 2A) and Congo red binding fluorescence (Fig. 2B)22, enhancing the fluorescence of these two amyloid probes. All fibrils were also characterized by DOT blot by the use of the antibody LOC23, that recognizes an epitope common to amyloid fibrils (Fig. S5). The extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein-science protocols to their purification and detection3,24. We chose the western blot methodology to detect amyloid fibers due their high sensitivity. To detect amyloid fibrils by the western blot methodology, the polymeric fibrils must be depolymerized into monomers. To achieve

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this, we dissolved the fibrils in 8 M urea (pH 2.3, 2% SDS) before boiling and running on an SDSPAGE gel. Using this protocol, we were able to monomerize alpha-synuclein (Fig. 2F), gelsolin fibrils (Fig. 2G) and Aβ1–40 fibrils (Fig. 2H), even though dimers and trimers were still present, and we detected low concentrations (0.3 µM) of all proteins by western blotting.

Figure 2. Characterization of amyloid fibrils of alpha-synuclein and gelsolin. (A) Emission spectra of 65 µg/ml amyloid fibrils of alpha-synuclein, gelsolin and Aβ1–40 incubated in PBS with

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0.3 µM p-FTAA. The spectra were collected at 25 °C with excitation at 450 nm. As a negative control, no protein was added in to the cuvette (buffer). (B) All fibrils were incubated with 10 µM Congo red and visualized by fluorescence microscopy using the red channel. Transmission electron microcopy of alpha-synuclein (C), gelsolin (D) and Aβ1–40 (E) amyloid fibrils. The amyloid fibrils from alpha-synuclein (F), gelsolin (G) and Aβ1–40 (H) at 10 µM were incubated in 8 M urea (pH 2.3, 2% SDS) and sonicated for 30 min in order to disrupt the fibrils into monomeric species. The samples were serially diluted to 5, 2.5, 1.25, 0.6 and 0.3 µM before boiling in sample buffer and then used for SDS-PAGE. Western blotting using specific antibodies was performed and visualized by infrared light.

The next step was the development of a pull-down protocol to isolate amyloid fibrils through the use of p-FTAA-R (Fig. 3A). Before incubation, aliquots of the samples were removed and denominated load (L) (Fig. 3A). The protocol consists of incubation of a solution containing amyloid fibrils, and the resin, for 24 h with rotation at 25 °C. Then, the resin was pelleted by centrifugation (step 1, 100 x g for 1 min at 15 °C) and the removed supernatant was called non-bound (step 2, NB). The resin was washed with one volume of phosphate-buffered saline (PBS) for 5 min and precipitated as described previously. The recovered supernatant was called wash 1 (step 3, W1) and this step was repeated twice (W2 and W3). Finally, the resin was resuspended in one volume of 8 M urea (pH 2.3, 2% SDS) with rotation for 24 h, in order to perform a denaturation of any amyloid fibrils bound to the resin (step 4). The resin was precipitated by centrifugation and the supernatant was called eluate (E) (step 5, Fig. 3A). Each sample (L, NB, W1, W2, W3 and E) was incubated with 8 M urea (2% SDS) and boiled for 10 min with Laemmli sample buffer before SDS-PAGE and western blot detection (Fig. 3B and 3C). Using the protocol described earlier, we compared the affinity of p-

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FTAA-R for the protein alpha-synuclein in different conformations, namely, monomeric or polymeric amyloid fibrils (Fig. 3B and 3C). For monomers, we observed that most of the protein was present in the non-bound fraction (NB). A small fraction was also present in W1, but no protein was detected in the subsequent fractions (W2, W3 and E) indicating that p-FTAA-R has no affinity for the monomeric alpha-synuclein (Fig. 3B). When the same protocol was applied to alphasynuclein amyloid fibrils, most of the protein was present in the eluate (E) fraction and some protein was also found in the non-bound (NB) and wash (W1) fractions (Fig. 3C). We concluded that pFTAA-R can distinguish between the monomeric versus the polymeric amyloid confirmations of alpha-synuclein.

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Figure 3. The pull-down protocol with p-FTAA-R discriminates between amyloid fibrils and monomeric species of alpha-synuclein. (A) Experimental scheme of the pull-down protocol. Western blot analyses of monomeric (B) or amyloid fibrils (C) of alpha-synuclein submitted to the protocol described in panel A. The band intensity was quantified and normalized in relation to the load (L 100%). The standard deviation of two independent experiments is showed.

To verify the threshold sensitivity of our protocol, we incubated the p-FTAA-R with decreasing concentrations of alpha-synuclein fibrils (Fig. S6). We observed that the p-FTAA-R was able to pull down amyloid aggregates of alpha-synuclein at 5, 2.5, 1, 0.5 and 0.25 µM (Fig. S6A and S6B). The amount of protein recovered in the eluate fraction was linear in this range of concentration (Fig. S6C). The experiments described in Figure 3 and Figure S6 were performed in phosphate-buffered saline (PBS) and we asked if p-FTAA-R could pull down amyloid fibrils in a complex solution of proteins. To address this point, we incubated 1 µM (70 µg/ml) of alpha-synuclein fibrils in human plasma and performed the pull-down protocol (Fig. 3A). The samples were divided in two gels, one to measure the total protein content (silver staining) and the other to detect alpha-synuclein (western blot) (Fig. 4). The protein concentration of human plasma is 60 g/l, so the fibrils represent less than 0.1% of the total protein concentration. When the silver gel was analyzed, we observed that almost all proteins in plasma were present in the non-bound (NB) fraction (Fig. 4A). On the other hand, the majority of alpha-synuclein was found in the eluate (E) fraction (Fig. 4B), similar to when the experiment was conducted in PBS (Fig. 3B). We concluded that p-FTAA-R was very selective since it was able to pull down the amyloid fibril of alpha-synuclein (Fig. 3C and Fig. 4B) but no monomeric alpha-

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synuclein (Fig. 3B) or other plasma protein (Fig. 4A). Moreover, the p-FTAA was also sensitive since it captured alpha-synuclein fibrils that comprised just 0.1% of the load (Fig. 4).

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Western blot for alpha-synuclein Figure 4. Pull down of alpha-synuclein fibrils added into plasma. Recombinant alpha-synuclein fibrils (1 µM) were added into human plasma and the sample submitted to the protocol described in panel A of the Figure 3. After denaturation (8M urea, 2% SDS, pH 2.3 and 10 min boilng) the samples were analyzed by silver staining (A) or western blot for alpha-synuclein (B). The band

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intensity was quantified and normalized in relation to the load (L 100%). The standard deviation of two independent experiments is showed.

To learn if p-FTAA-R could be used for general amyloid purification, we repeated the pull-down experiments using Aβ1–40 and gelsolin amyloid fibrils (Fig. 5 and S7). In both cases, the resin was efficient in capturing the amyloid fibrils (Fig. 5A and Fig. S7), as described before for alphasynuclein fibrils (Fig. 3C). It is important to note that the eluate sample possessed low amounts of protein when compared to the load for all fibrils tested (compare E with L in Fig. 3C, 5A and S7). It can be partially explained, because a certain amount of the fibrils does not bind to the resin (Fig. 3C). However, for gelsolin fibrils this is not the case (Fig. 5A). We speculate that upon binding to pPFTAA-R, the stability of the fibrils increased, making the elution process more difficult, even in the presence of 8 M urea (pH 2.3 and 2% SDS). To address whether some amyloid fibrils could still be trapped in the p-FTAA-R after the elution step (step 5, Fig. 3A), we applied to a nitrocellulose membrane the p-FTAA-R that were recovered by centrifugation after treatment with 8 M urea (pH 2.3 and 2% SDS) for 24 h. In fact, we detected the signal of gelsolin bound to p-FTAA-R by DOTblot (data not shown) confirming that the elution process was not sufficient to denature the fibers attached to the p-FTAA-R. Another observation that points in this direction is the enhanced detection of dimers, trimers and tetramers of gelsolin in the elution fraction when compared with the load (Fig. 5A), suggesting higher stability of the gelsolin-bound fibrils. As a negative control for the experiments described in Figure 5A and Figure S7 we used an agarose resin where no coupling had been performed (nc-R) since amyloid fibrils are not soluble species and our protocol involves centrifugation steps (Fig. 3A). We observed that no amyloid fibril binding occurred when nc-R was used for pulling down gelsolin (Fig. S8) and Aβ1–40 fibrils (Fig. S7), indicating that the presence of

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p-FTAA is essential for the success of the protocol. We also included as a control the use of a nonamyloid aggregate. We incubated the protein alpha-synuclein at pH 4.0, 37 C° for 24 hs, a protocol that induces the formation of amorphous aggregates25. We confirmed the absence of fibrils or oligomers by TEM morphology inspection and by the thioflavin T binding (Fig. S9A and 9B). Then, we performed the pull-down protocol described in Figure 3A with amorphous aggregates of alphasynuclein and the p-FTAA-R. We observed that most of the sample was present in the non-bound fraction (Fig. S9C) confirming that our protocol is specific to amyloid aggregates. Next, we used in vivo amyloid fibrils extracted from the muscles of a mouse model of amyloidosis26. Transgenic mouse models of FAF possess the insertion of human gelsolin D187N variant (83 kDa) that after aberrant cleavage by furin in the trans-Golgi (68 kDa) and further cleavage in the extracellular space affords the 8 kDa FAF-associated amyloidogenic fragments in dermis and muscles26. Histochemistry revealed that a cutaneous muscle fibril presents amyloid deposition as revealed by p-FTAA fluorescence (Fig. 5B). Total protein extracted from the muscles of 18-month-old D187N mice were subjected to the pull-down protocol with p-FTAA-R. As a negative control, we used extracts from wt muscles and observed that the load fraction from a D187N mouse presented gelsolin bands (83, 68 and 8 kDa) that were absent in wt extracts (Fig. 5C compare L with wt). Using just the transgenic muscle extracts and the protocol described in Figure 3A, we observed that a certain amount of gelsolin was present in the non-bound fraction, but most of the protein was detected in the eluate (Fig. 5C). Interestingly, as seen with in vitro gelsolin fibrils (Fig. 3A), we observed dimers, trimers and high molecular weight (HMW) species of gelsolin in the eluate fraction of in vivo fibrils (Fig. 5C).

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Figure 5. In vitro and in vivo pull-downs of gelsolin amyloid fibrils. (A) Recombinant gelsolin amyloid fibrils were submitted to the pull-down protocol (Fig. 3A) and analyzed by western blot. (B) Fluorescence image of amyloid deposits in tissue of gelsolin transgenic mice stained with pFTAA. The nuclei of the cells were stained with DAPI. (C) Muscle extracts from gelsolin transgenic mice were submitted to the pull-down protocol described in Figure 3A. The samples were analyzed by western blot using the gelsolin antibody. As negative control, muscle extract from wild-type mice was applied in the second lane (wt) of the gel. Species with molecular mass above 100 kDa are denominated high molecular weight (HMW) species. The band intensity was quantified and normalized in relation to the load (L 100%). The standard deviation of two independent experiments is showed.

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Finally, we asked if the p-FTAA-R could be able to pull-down oligomeric species that precede amyloid polymerization. To address this point, we purified the variant A30P of alpha-synuclein, a mutation that induces the formation of oligomers27. The oligomers of A30P were purified and then characterized by different techniques: dot-blot with the antibody A11, that is selective for oligomers28 (Fig. 6A); TEM (Fig. 6B); fluorescence spectroscopy with the Bis-ANS probe that shows high affinity to oligomers (Fig. 6C) and thioflavin T probe that shows low biding to oligomers (Fig. 6D) 29. Interesting, the p-FTAA-R was efficient in pulling down A30P oligomers added in PBS (Fig. 6E). Our protocol requires an incubation of 24hs of the sample with the resin (step 1, Fig. 3A), so, we cannot exclude the possibility that A30P oligomers evolved to mature amyloid fibrils during this time. However, this seems unlikely since the oligomers were added at low concentration (5 µM) and the amyloid formation of alpha-synuclein usually occurs above 70 µM.

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Figure 6. Pull-down of oligomers of alpha-synuclein. (A) DOT blot of 5 µM of monomer and oligomers of A30P alpha-synuclein against the conformational oligomer antibody (A11 Ab). Alphasynuclein antibody (α-syn Ab) was used as a load control. (B) Transmission electron microscopy of A30P oligomers. Bis-ANS (C) and thioflavin T (D) fluorescence of 5 µM oligomers or amyloid fibrils of A30P. For Bis-ANS excitation was 360 nm and emission 400-600 nm while for thioflavin T excitation was 450 nm and emission 470-520 nm. The extent of Bis-ANS and thioflavin T binding was evaluated by measuring the spectral area. (E) Purified A30P oligomers were submitted to the pull-down protocol (Fig. 3A) and analyzed by western blot. The band intensity was quantified and

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normalized in relation to the initial load (L 100%). The standard deviation of two independent experiments is showed.

CONCLUSION In conclusion, p-FTAA was successfully introduced in the fabrication of an agarose resin for achieving the purification of amyloid fibrils from complex solutions. P-FTAA-R exhibits selectivity and sensitivity towards the amyloid fold. By combining the pull-down protocol with western blot detection the amyloid fibrils produced in vitro from three different proteins were purified. In vivo fibrils from FAF mouse models were efficiently purified and detected from muscle tissue extracts, demonstrating the feasibility of p-FTAA-R for the diagnosis of amyloid disorders and for the identification of new examples of functional amyloids.

MATERIAL AND METHODS Pentameric thiophene synthesis p-FTAA was synthesized as previously described13. Coupling of pentameric thiophene in to agarose resin Four ml of agarose CarboxyLink Coupling Gel Immobilized Diaminodipropylamine (Thermo Scientific) was equilibrated with coupling buffer (0.1M MES buffer [2-(NMorpholino)ethansulfonic acid], 0.9% NaCl, pH 4.7. Five mg of p-PFTAA was solubilized into 100% DMSO diluted in 2 ml coupling buffer and mixed with the agarose resin. Sixty mg of [1Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl] (EDC) was solved in 0.5 ml of coupling buffer

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and immediately mixed with the resin + p-FTAA achieving the concentration of 70 mM. The mixture (resin + p-FTAA + EDC) was incubated with agitation for 3 hs at 25 °C. The resin was extensively washed with 1M NaCl and phosphate saline buffer (PBS) until the absence of detection of p-FTAA by absorbance at 450 nm into flow through. Preparation of amyloid fibrils Alpha-synuclein30, 8 kDa gelsolin fragment (residues 173-242) 31 and Aβ1-4032 were purified as previously described. Aβ1-40 at 216 µg/ml was aggregated in 50 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 0.02% NaN3 at 37 °C for 7 days with agitation. Alpha-synuclien at 1,960 µg/ml was incubated in 10 mM Tris buffer pH 7.4, 100 mM NaCl, 0.02% NaN3 under the same conditions described for Aβ1-40. The 8 kDa gelsolin fragment peptide was incubated at 60 µg/ml in 50 mM sodium phosphate buffer, pH 6.8, 100 mM NaCl, 0.02% NaN3 at 37 °C with agitation for 24 h. The sample was then centrifuged (16,000 g for 15 min at 4 °C) and the pellet was resuspended in 50 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 0.02% NaN3 to obtain a final concentration of 600 µg/ml. Preparation of amorphous aggregates Purified alpha-synuclein (500 µM) was incubated with 50 mM sodium acetate (pH 4.0) for 24 hs with agitation as described previoulsy25. Oligomers purification The A30P alpha-syncuclein oligomers were purified as described previously33. Congo red, p-FTAA and Bis-ANS binding assays.

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Fibril formation was assessed using Congo red and p-FTAA binding assays. For Congo red binding, the samples were diluted to a final concentration of 65 µg/ml in 5 mM potassium phosphate and 150 mM NaCl at pH 7.4 containing 10 µM Congo red and fluorescence was recorded at red channel in a fluorescence microcopy at 400X magnification 22. For p-FTAA binding assays, the samples were diluted to 65 µg/mL in 5 mM potassium phosphate and 150 mM NaCl at pH 7.4 containing 0.3 µM p-FTAA and binding was monitored using a spectrofluorimeter to measure the fluorescence increase (excitation at 450 nm and fluorescence emission at 480-700 nm)13. For Bis-ANS binding assays, the samples (A30P oligomers or amyloid fibrils) were diluted to 5 µM in PBS containing 1 µM BisANS and binding was monitored using a spectrofluorimeter to measure the fluorescence increase (excitation at 360 nm and fluorescence emission at 400-600 nm) Western blotting The samples were sonicated for 30 min in denaturing buffer (8M urea, 2% SDS, pH 2.3) and boiled for 10 min in the presence of Laemmli buffer in order to monomerize the fibrils. SDS-PAGE was performed under reducing conditions. Samples were transferred to PVDF and probed with syn-1 antibody (1:10,000) for alpha-synuclein or a rabbit polyclonal antibody (1:10,000) developed by Balch’s group 26 for gelsolin. Blots were then probed with goat anti-mouse secondary antibody conjugated to IRDye 800CW (1:10,000) for alpha-synuclein and goat anti-rabbit secondary antibody conjugated to IRDye 800CW (1:10,000) for gelsolin and developed/quantified using an Odyssey Infrared Imaging System. Dot blot assay Samples were spotted (2 µl) onto nitrocellulose membrane. The membrane was blocked using 1vol PBS + 1vol blocking solution (Odyssey) for 1 h. The membrane was incubated with 6E10 antibody

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(1:10,000) for Aβ1-40, syn-1 antibody (1:10,000) for alpha-synuclein or a rabbit polyclonal antibody (1:10,000) for gelsolin and diluted in 1vol TBST (50 mM Tris pH 7.6, 0.9% NaCl, 0.1% Tween 20) + 1vol blocking solution for 1 h, washed 3 times with TBST and then incubated for 1 h with then probed with goat anti-mouse (Aβ1-40, syn-1) or anti-habit (gelsolin) secondary antibody conjugated to IRDye 800CW (1:10,000) and developed/quantified using an Odyssey Infrared Imaging System. LOC23 antibody can detect amyloid fibrils while A1128 oligomeric species. The membrane was incubated with LOC antibody (1:1,000, Millipore) or A11 (1:1,000, Sigma) (diluted in 1 vol TBST +1 vol blocking solution for 1 h, washed 3 times with TBST and then incubated for 1 h with goat anti-rabbit secondary antibody conjugated to IRDye 800 CW (1:5,000) and developed/quantified using an Odyssey Infrared Imaging System. Plasma sample Human plasma was purchased from Biochemed Services, Winchester, VA and stored at -80 °C until use. Scanning Electron Microscopy The resins were washed with distilled water and allowed to adhere to 1% gelatin-coated glass coverslips. Next the samples were dehydrated in ethanol, critical point dried with liquid CO2, coated with carbon and then observed with a JEOL 5600 scanning electron microscope operating at 15KV in low vacuum mode. Immunohistochemistry. Mice tissue samples were diagnosed as positive for amyloidosis using p-FTAA staining13. Tissues were deparaffinized using two washes of 5 min in 100% xylene and 3 washes of 5 min in ethanol

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(100%, 90% and 70%), and 5 min in dH2O. The samples were incubated with 0.3µM p-FTAA and DAPI (10 µg/mL; Sigma) for 30 min and then rinsed with PBS. Images were taken using a 40x oil-immersion objective in a Leica TCS SP5 II confocal microscope (Leica Microsystems). For image acquisition the software LAS AF Lite (Leica Microsystems) was used and images were merged using Adobe Photoshop 7.0 (Adobe). Transmission Electron Microscopy The samples were prepared as described by Azevedo and colleagues 22. Western blot quantification The western blot quantification was performed by the software Fiji34. Protein quantification Total protein concentrations were determined using the Pierce BCA assay according with manufacturers’ instructions (Pierce). Preparation of muscle extracts Homozygous D187N (+/+) C57BL/6J mice were sacrificed at 18 months of age26. Mice were saline perfused and isolated tissues homogenized in PBS containing protease inhibitors (Roche) by using a Teflon mechanical homogenizer at 70 rpm and centrifuged at 1,000 g for 3 min at 4 °C to obtain post debris supernatant, as previously described 26. As negative control we used wild type C57BL/6J mice. Protein concentrations were determined using BCA and equal amounts of D187N (+/+) or wt extracts were loaded onto SDS/PAGE gels. All animal procedures complied with Brazilian legislation and were reviewed and approved by the Ethics Committee for Animal Experimentation (CEUA) of the Federal University of Rio de Janeiro.

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Pull down protocol Five hundred microliters of sample was incubated with 50 µl of resin at 25 °C (except for experiments preformed with tissue extract where the temperature was 4 °C). All samples were incubated with 0.02% sodium azide to avoid any bacterial growth. Before incubation with the resin 50 µl of each sample was removed and stored at -20 °C as load (L). After incubation with rotation over night the sample was centrifuged (100 x g, 1 min at 15 °C) and the supernatant removed (non bound). The resin was ressuspended with 500 µl PBS and incubated for 5 min (wash 1) before centrifugation (100 x g, 1 min at 15 °C). The wash step was repeated two more times (wash 2 and 3). The resin was ressuspended with 500 µl of denaturation buffer (8M urea, 2% SDS, pH 2.3) and incubated over night with agitation at 25 °C. After centrifugation the supernatant was removed (eluate). For the experiment described in Figure 3 the concentration of monomeric of fibrilar alphasynuclein was 5 µM. For the experiment with plasma the concentration of fibrilar alpha-synuclein was 1 µM. For the experiments in Figures 5A and S6 the gelsolin concentration used was 3 µM. The Aβ1-40 fibril concentration used in Figure S4 was 15 µM. The total protein concentration of muscle extracts (Figure 5C) used was 5 mg/ml.

ACKNOWLEDGMENTS We thank Wanderley de Souza, Yraima Cordeiro, Russolina Zingali and Eleonora Kurtenbach for the use of laboratory facilities. We also thank Lesley Page and William Balch who provided us with the gelsolin transgenic mice tissues and gelsolin antibody. We thank Fabio Almeida and Tatiana Domitrovic for the critical reading of the manuscript, Jeffery Kelly, Neil Grimster, Aleksandra Baranczak and Debora Foguel for helpful suggestions and Santiago Alonso and Samantha Seifert for technical support.

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CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

AUTHOR CONTRIBUTIONS ABW and LF contributed equally to this work. FLP conceived and coordinated the study and wrote the paper. FLP, ABW, LF and MK designed, performed and analyzed the experiments shown in Figures 1-6. AP designed, performed and analyzed the experiments shown in Figure 1F and 1G. CSM and DRR were responsible for p-FTAA synthesis and characterization. All authors reviewed the results and approved the final version of the manuscript.

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Table of Contents/Abstract Graphic Pentathiophene Resin linker

Resin

Amyloid fibril

Agarose resin

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ABSTRACT Protein aggregation is a hallmark of several degenerative

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