Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

  Citation: Ferreira, E.; Almeida, Z.L.; Cruz, P.F.; Silva e Sousa, M.; Veríssimo, P.; Brito, R.M.M. Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption. Int. J. Mol. Sci. 2022,23, 391. https://doi.org/ 10.3390/ijms23010391 Academic Editor: Vytautas Smirnovas Received: 31 August 2021 Accepted: 20 December 2021 Published: 30 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption Elisabete Ferreira 1,†, Zaida L. Almeida 1,† , Pedro F. Cruz 1, Marta Silva e Sousa 1, Paula Veríssimo 2 and Rui M. M. Brito 1,* 1Chemistry Department and Coimbra Chemistry Centre-Institute of Molecular Sciences (CQC-IMS), University of Coimbra, 3004-535 Coimbra, Portugal; [email protected] (E.F.); [email protected] (Z.L.A.); [email protected] (P.F.C.); [email protected] (M.S.e.S.) 2Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; [email protected] *Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies—amyloidoses—are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented. Keywords: transthyretin; protein aggregation; amyloid fibrils; amyloidosis; amyloid disruptors; fibril disaggregation; screening protocol 1. Introduction Currently, considerable attention is given to a group of protein misfolding diseases known as amyloidosis [ 1 – 4 ]. There are approximately 50 of these pathologies, involving different precursor proteins and associated with the formation of extracellular amyloid fibrils or intracellular inclusions with amyloid-like characteristics [ 3 , 5 ]. Consequently, amyloidoses can affect different organs, namely the heart, liver, kidneys, nervous system, spleen, skin, and gastrointestinal tract, among others. Severe amyloidosis can lead to life-threatening organ failure and death. No effective disease modifying therapies are available for most amyloid diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Amyloidβ peptide, prion protein, α -synuclein, and transthyretin (TTR) are examples of the more than 40 different human peptides and proteins identified in amyloid deposits that are at the origin of the known amyloidoses [3]. Interestingly, amyloid fibrils present common morphologies and structural properties, despite being originated by quite different precursor proteins. Amyloid fibrils are long unbranched structures with a high β -sheet content structural core and thermodynamically Int. J. Mol. Sci. 2022,23, 391. https://doi.org/10.3390/ijms23010391 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 391 2 of 17 very stable, which may be detected through binding to dyes, such as Congo red, or the fluorescent probe thioflavin-T (ThT) [1,6]. Amyloids formed by the protein transthyretin (TTR) are at the origin of several amyloidoses (ATTR) that can be divided in two main types: sporadic, age-related amyloidosis, and hereditary or familial amyloidosis. Wild-type TTR amyloidosis (ATTRwt), formerly known as senile systemic amyloidosis (SSA), is an idiopathic disease characterized by the deposition of wild-type (wt) TTR amyloid mainly in the heart. ATTRwt is strongly correlated with ageing, affecting an estimated 25% of the world population over the age of 80 [ 7 ], and may give rise to heavy deposition and infiltration of amyloid in the cardiac tissue, resulting in congestive heart failure [ 8 ]. Mutant TTR amyloidosis (ATTRm) comprises a group of familial/hereditary and rare multisystem diseases with a wide spectrum of clinical manifestations, such as familial amyloid polyneuropathy (hATTR-PN or FAP) [ 9 ], familial amyloid cardiomyopathy (hATTR-CM or FAC) [ 10 ], and familial leptomeningeal amyloidosis [ 11 ], which may occur in isolation or in combination with familial ocular amyloidosis [ 12 ] and additional clinical manifestations. These ATTRm amyloidoses are caused by more than 130 different known TTR variants [13,14]. Human TTR is a 55 kDa homotetrameric plasma protein with a high proportion of β -sheet structure that is mainly biosynthesized in the liver [ 15 ], but also synthesized in the choroid plexus of the brain [ 16 ] and in the retinal pigmentous epithelium of the eye [ 17 ]. The main known functions of TTR are the transport of thyroid hormones and retinol in association with retinol-binding protein. Additionally, TTR is also known to have a neuroprotective role [18]. Over the years, the molecular mechanisms of amyloid formation have been the subject of considerable scientific interest [ 19 , 20 ]. In vitro fibril formation has been a powerful tool in the study of the mechanism that leads to amyloid formation, helping to explain the events occurring in vivo . Regarding the TTR aggregation process, TTR instability caused by mutations or by other factors, leads to the dissociation of the native tetramer into monomers that undergo partial unfolding [ 21 – 23 ]. These non-native monomers can associate into soluble oligomers that self-assemble, leading to the final amyloid fibril [ 20 , 24 ]. However, the clinical and genetic heterogeneity of ATTR, together with the different structural and functional characteristics of the TTR variants, suggests that TTR fibril formation may involve diverse molecular pathways [ 25 ]. Amyloid deposits may be constituted not only by full-length TTR fibrils, but also by C-terminal TTR fragments and amorphous aggregates [26–28]. Although liver transplantation and therapies [ 29 , 30 ] such as TTR stabilization [ 27 ] or suppression of TTR expression [ 31 – 36 ] are currently in use, there is no fully effective treatment for all the clinical manifestations of ATTR amyloidosis yet. Considering the current knowledge of TTR and TTR amyloid formation, various therapeutic strategies targeting different steps of the aggregation pathway may be devised [ 28 – 30 ]. The disruption of amyloid aggregates and fibrils is one of these strategies, and has gained importance in both experimental assays and clinical studies [ 37 – 46 ] in the last two decades. However, to date, only a few compounds have been identified as possible amyloid fibril disruptors for ATTR. Among these are some polyphenols, such as EGCG (epigallocatechin-3-gallate) [37,38,45] , IDOX (40-iodo-40-deoxydoxorubicin) [39,40], and doxycycline [42,43,46]. In order to discover new amyloid fibril disruptors against ATTR, we aimed to develop an appropriate in vitro screening protocol for TTRwt disaggregation. For this purpose, the first step was the characterization and selection of an appropriate aggregation protocol and identification of the respective amyloid aggregate/fibril model. Ideally, the in vitro formed amyloid species would have similar structure and morphology to in vivo formed species, and the aggregation protocol experimental conditions would be as close as possible to the in vivo process of TTR amyloid formation, maintaining physiological conditions, such as average protein concentration, pH, ionic strength, and temperature. However, the native tetrameric form of TTRwt has high conformational stability [ 47 – 49 ], and the amyloid formation process in these conditions is lengthy and thus is unsuitable to be used in screening Int. J. Mol. Sci. 2022,23, 391 3 of 17 protocols. After a careful analysis of the available literature [ 21 , 50 , 51 ], three aggregate and fibril formation protocols were selected for comparison: acidification at pH 2.0 and pH 4.4, and heating at pH 7.4. Furthermore, the effect of changing the pH from 2.0 or 4.4 to physiological conditions (pH 7.4), after aggregation, was also studied. Thioflavin-T (ThT) fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to characterise the timing and products formed with these protocols and the effect of pH modification. The choice of appropriate experimental conditions and methods to provide relevant information on the effect of particular compounds on fibril interaction and disaggregation is of paramount importance in a screening protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was used to study the interaction of TTR fibrillar structures and a known TTR fibril disruptor—doxycycline—in more detail. DLS, TEM, and turbidity experiments were used to characterize the effect of this compound on TTR amyloid fibril disruption. Additionally, doxycycline was chosen to validate the experimental protocols due to its well demonstrated anti-amyloidogenic activity in vitro [ 46 ] and in vivo . It has been shown that doxycycline acts as L55P TTR amyloid fibril disruptor in vitro [ 40 ] and disaggregates amyloid deposits in V30M TTR transgenic mice with a concomitant decrease of various amyloidosis tissue markers [ 41 , 42 ]. This compound was even used in clinical trials, sometimes in combination with TUDCA (tauroursodeoxycholic acid) [ 43 ] and other times in combination with UDCA (ursodeoxycholic acid) [52]. 2. Results 2.1. Characterization of TTRwt Fibril Formation Protocols In order to summarize the three aggregation protocols of TTRwt used in this study, Table 1shows the main requirements for these protocols in terms of experimental conditions, and Table 2summarizes the main characteristics of the aggregates and fibrils formed during aggregation and the result of pH changes on these amyloid structures. Table 1. Experimental conditions of three TTRwt aggregation protocols. pH Temperature Incubation Period Protein Concentration Stirring 2 25 ◦C At least 1 week 80 µM and then diluted to 3.6 µMNo 4.4 37 ◦C 3 days 3.6 µM No 7.4 60 ◦C 6 days 3.6 µM No Table 2. Characterization of TTRwt amyloid species formed by different aggregation protocols. Aggregation Protocol Morphology of the Aggregates and Fibrils Length/Diameter of Aggregated Species Thioflavin-T Assay Effect of pH Adjustment to pH 7.4 pH 2.0 Long, unbranched, mature fibrils 50 to 170 nm/5 to 9 nm Positive Alterations in the size and secondary structure of fibrils Fibrils remain ThT positive pH 4.4 Mixture of spheroid structures, amorphous aggregates, and short unbranched fibrils Spheroid aggregates (4 to 6 nm); fibrils (25 nm/4–5 nm) Positive Slight effect on size, secondary structure, and morphology of aggregates to more amorphous and less fibrillar species Fibrils remain ThT positive pH 7.4 Unbranched fibrils 10 to 50 nm/4 to 6 nm Positive Not necessary According to Tables 1and 2, the fibril formation protocol performed at pH 2.0 with 0.1 M NaCl was more time-consuming and required higher concentrations of protein. Amyloid fibrils observed by TEM appeared as long unbranched filamentous structures 5 to 9 nm in diameter with variable length, indicating a mature growth of these fibrillar Int. J. Mol. Sci. 2022,23, 391 4 of 17 structures [ 1 ] which were ThT positive. The far-UV CD spectrum also showed a minimum around 213 nm, and a CD spectrum typical of TTRwt amyloid fibrils [ 20 ] with high β -sheet content (Table 3). In an attempt to mimic the in vivo conditions of TTRwt amyloid fibrils, pH adjustment to pH 7.4 of preformed fibrils at pH 2.0 was carried out, and the effect on fibril size, structure, and morphology was analysed by DLS, CD, and TEM, respectively. DLS results showed that after 11 days of fibril incubation at pH 7.4, the sample had larger populations of smaller particles (between 100 and 1000 nm) compared to fibril incubation at pH 2.0 (Figure S1). The changes in secondary structure of preformed TTRwt fibrils were analysed by far-UV CD and the minimum at 213 nm was not as pronounced, indicating a clear loss in β -sheet and a gain of unordered secondary structures (Figure 1A and Table 3). TEM observations indicated that, at pH 7.4, fibrils lost their extended fibrillar structure and became more spherical and amorphous with diameters between 4 and 13 nm when compared to fibrils at pH 2.0 (Figure 2A, pH 2.0 to 7.4), corroborating DLS and CD results; however, they still remained ThT positive. Table 3. Secondary structure estimation by Circular Dichroism using the web server BeStSel. Aggregation Protocol pH 2.0 pH 2.0 → pH 7.4 pH 4.4 pH 4.4 → pH 7.4 pH 7.4 (60 ◦C) pH 7.4 (37 ◦C) α-Helix 0.14 0.03 0.07 0.08 0.11 0.09 β-Sheet 0.43 0.32 0.32 0.24 0.4 0.39 Turn 0.09 0.13 0.16 0.17 0.11 0.13 Unordered 0.34 0.52 0.45 0.51 0.38 0.39 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 17 fibrils amorphous and less fibrillar species pH 7.4 Unbranched fibrils 10 to 50 nm/4 to 6 nm Positive Not necessary According to Tables 1 and 2, the fibril formation protocol performed at pH 2.0 with 0.1 M NaCl was more time-consuming and required higher concentrations of protein. Amyloid fibrils observed by TEM appeared as long unbranched filamentous structures 5 to 9 nm in diameter with variable length, indicating a mature growth of these fibrillar structures [1] which were ThT positive. The far-UV CD spectrum also showed a minimum around 213 nm, and a CD spectrum typical of TTRwt amyloid fibrils [20] with high βsheet content (Table 3). In an attempt to mimic the in vivo conditions of TTRwt amyloid fibrils, pH adjustment to pH 7.4 of preformed fibrils at pH 2.0 was carried out, and the effect on fibril size, structure, and morphology was analysed by DLS, CD, and TEM, respectively. DLS results showed that after 11 days of fibril incubation at pH 7.4, the sample had larger populations of smaller particles (between 100 and 1000 nm) compared to fibril incubation at pH 2.0 (Figure S1). The changes in secondary structure of preformed TTRwt fibrils were analysed by far-UV CD and the minimum at 213 nm was not as pronounced, indicating a clear loss in β-sheet and a gain of unordered secondary structures (Figure 1A and Table 3). TEM observations indicated that, at pH 7.4, fibrils lost their extended fibrillar structure and became more spherical and amorphous with diameters between 4 and 13 nm when compared to fibrils at pH 2.0 (Figure 2A, pH 2.0 to 7.4), corroborating DLS and CD results; however, they still remained ThT positive. Table 3. Secondary structure estimation by Circular Dichroism using the web server BeStSel. Aggregation Protocol pH 2.0 pH 2.0 → pH 7.4 pH 4.4 pH 4.4 → pH 7.4 pH 7.4 (60 °C) pH 7.4 (37 °C) α-Helix 0.14 0.03 0.07 0.08 0.11 0.09 β-Sheet 0.43 0.32 0.32 0.24 0.4 0.39 Turn 0.09 0.13 0.16 0.17 0.11 0.13 Unordered 0.34 0.52 0.45 0.51 0.38 0.39 Figure 1. Far-UV CD spectra of TTRwt fibrils from different fibril formation protocols. (A) TTRwt fibrils assembled at pH 2.0 and after pH adjustment to 7.4, (B) at pH 4.4 and after pH adjustment to 7.4, and (C) at pH 7.4 at 60 °C and after temperature change to 37 °C. All TTRwt samples were analysed at 3.6 μM and all CD spectra were acquired at the end of each fibril formation protocol. Regarding the aggregation at pH 4.4, this protocol was the least time-consuming, had low protein requirements, and was suitable for pH adjustment to 7.4 (Tables 1 and 2). The TEM images of the amyloid structures formed showed shorter fibrils that were 5 to 6 nm in diameter and at least 25 nm in length, as well as some amorphous aggregates and several oligomeric species that were 4 to 6 nm in diameter (Figure 2A, pH 4.4). The CD spectrum also indicated a less pronounced β-sheet profile, corroborating the TEM results (Figure 1B and Table 3). In addition, the DLS and CD experiments showed that pH adjustment Figure 1. Far-UV CD spectra of TTRwt fibrils from different fibril formation protocols. ( A ) TTRwt fibrils assembled at pH 2.0 and after pH adjustment to 7.4, ( B ) at pH 4.4 and after pH adjustment to 7.4, and ( C ) at pH 7.4 at 60 ◦ C and after temperature change to 37 ◦ C. All TTRwt samples were analysed at 3.6 µM and all CD spectra were acquired at the end of each fibril formation protocol. Regarding the aggregation at pH 4.4, this protocol was the least time-consuming, had low protein requirements, and was suitable for pH adjustment to 7.4 (Tables 1and 2). The TEM images of the amyloid structures formed showed shorter fibrils that were 5 to 6 nm in diameter and at least 25 nm in length, as well as some amorphous aggregates and several oligomeric species that were 4 to 6 nm in diameter (Figure 2A, pH 4.4). The CD spectrum also indicated a less pronounced β -sheet profile, corroborating the TEM results (Figure 1B and Table 3). In addition, the DLS and CD experiments showed that pH adjustment to 7.4 slightly modified the particle size (Figures S2 and 2B, pH 4.4 to 7.4) and secondary structure (Figure 1B). The TEM images revealed more spheroid and amorphous morphologies and less fibrillar structures (4 to 5 nm in diameter, and up to 25 nm in length), which is in total agreement with a small loss of β -sheet structure and an increase in unordered structure (Table 3) when compared with the fibrils formed at 4.4, and even at pH 2.0 (Figure 2A, pH 4.4 to 7.4); however, they remained ThT positive (Table 2). Int. J. Mol. Sci. 2022,23, 391 5 of 17 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 17 to 7.4 slightly modified the particle size (Figures S2 and 2B, pH 4.4 to 7.4) and secondary structure (Figure 1B). The TEM images revealed more spheroid and amorphous morphologies and less fibrillar structures (4 to 5 nm in diameter, and up to 25 nm in length), which is in total agreement with a small loss of β-sheet structure and an increase in unordered structure (Table 3) when compared with the fibrils formed at 4.4, and even at pH 2.0 (Figure 2A, pH 4.4 to 7.4); however, they remained ThT positive (Table 2). Figure 2. Analysis of the effect of doxycycline on preformed TTRwt aggregates and fibrils. TTRwt amyloid species (3.6 μM) assembled at pH 2.0 (pH 2.0), pH 2.0 followed by pH adjustment to 7.4 (pH 2.0 to 7.4), pH 4.4 (pH 4.4), pH 4.4 followed by pH adjustment to 7.4 (pH 4.4 to 7.4), and induced by heating at pH 7.4 (pH 7.4) were incubated both in the absence (control) and presence of 50× molar excess of doxycycline (180 μM) for the indicated period of time and analysed by (A) TEM and (B) DLS. The scale bars represent 200 nm, and the arrows indicate some of the amyloid structures observed. Figure 2. Analysis of the effect of doxycycline on preformed TTRwt aggregates and fibrils. TTRwt amyloid species (3.6 µ M) assembled at pH 2.0 (pH 2.0), pH 2.0 followed by pH adjustment to 7.4 (pH 2.0 to 7.4), pH 4.4 (pH 4.4), pH 4.4 followed by pH adjustment to 7.4 (pH 4.4 to 7.4), and induced by heating at pH 7.4 (pH 7.4) were incubated both in the absence (control) and presence of 50 × molar excess of doxycycline (180 µ M) for the indicated period of time and analysed by ( A ) TEM and ( B ) DLS. The scale bars represent 200 nm, and the arrows indicate some of the amyloid structures observed. Regarding the heat-induced fibril formation at pH 7.4, this protocol had a duration of 6 days and required low concentrations of protein (Table 1). The resulting species were ThT positive (Table 2), and TEM images showed unbranched fibrillar structures that were shorter and narrower than the fibrils formed at pH 2.0, with a diameter of 4 to 6 nm and a length of 10 to 50 nm (Figure 2A, pH 7.4). The CD spectrum also displayed the typical β -sheet profile (Figure 1C and Table 3). To better characterize this protocol, turbidity was also used to follow the aggregation process. An increase in turbidity was observed in the 6 days of heating (Figure S3), which implies aggregation. The transition from 60 ◦ C to 37 ◦ C did not induce a decrease in turbidity, indicating that the decrease of temperature Int. J. Mol. Sci. 2022,23, 391 6 of 17 neither causes fibril breakdown (Figure S3, 8th day) nor an alteration in secondary structure (Figure 1C and Table 3). 2.2. Interaction of Doxycycline with Preformed TTRwt Fibrils The interaction between doxycycline (Figure 3A) and preformed TTRwt aggregates and/or fibrils, prepared according to the previously described aggregation protocols, was studied using 1 H STD NMR. The 1 H NMR spectrum of doxycycline (Figure 3B, light grey), 1 H STD NMR spectrum of doxycycline in the presence of TTRwt fibrils (Figure 3B, black), and the off-resonance spectrum (Figure 3B, grey) for each experimental condition is represented in Figure 3B. The signals present in the STD spectrum, identified by their chemical shifts, represent the doxycycline protons that are in close contact with TTR amyloid structures. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 17 Regarding the heat-induced fibril formation at pH 7.4, this protocol had a duration of 6 days and required low concentrations of protein (Table 1). The resulting species were ThT positive (Table 2), and TEM images showed unbranched fibrillar structures that were shorter and narrower than the fibrils formed at pH 2.0, with a diameter of 4 to 6 nm and a length of 10 to 50 nm (Figure 2A, pH 7.4). The CD spectrum also displayed the typical β-sheet profile (Figure 1C and Table 3). To better characterize this protocol, turbidity was also used to follow the aggregation process. An increase in turbidity was observed in the 6 days of heating (Figure S3), which implies aggregation. The transition from 60 °C to 37 °C did not induce a decrease in turbidity, indicating that the decrease of temperature neither causes fibril breakdown (Figure S3, 8th day) nor an alteration in secondary structure (Figure 1C and Table 3). 2.2. Interaction of Doxycycline with Preformed TTRwt Fibrils The interaction between doxycycline (Figure 3A) and preformed TTRwt aggregates and/or fibrils, prepared according to the previously described aggregation protocols, was studied using 1 H STD NMR. The 1 H NMR spectrum of doxycycline (Figure 3B, light grey), 1 H STD NMR spectrum of doxycycline in the presence of TTRwt fibrils (Figure 3B, black), and the off-resonance spectrum (Figure 3B, grey) for each experimental condition is represented in Figure 3B. The signals present in the STD spectrum, identified by their chemical shifts, represent the doxycycline protons that are in close contact with TTR amyloid structures. Figure 3. ( A ) Molecular structure of doxycycline and ( B ) 1 H STD NMR of doxycycline (2 mM) in the presence of preformed TTRwt fibrils (20 µ M) assembled at pH 2.0 (pH 2.0), pH 2.0 that underwent pH adjustment to 7.4 (pH 2.0 to 7.4), pH 4.4 (pH 4.4), and assembled by heating at pH 7.4 (pH 7.4). The reference 1 H NMR spectrum of doxycycline at the corresponding pH is represented in light grey, the 1 H STD NMR spectrum is represented in black, and the off-resonance spectrum is represented in grey. At pH 2.0, the doxycycline signals from the aromatic protons (i.e., H-15, H-16, and H-17), three methyl groups, and protons H-8, H-11, and H-4 were observed to interact with Int. J. Mol. Sci. 2022,23, 391 7 of 17 TTRwt amyloid fibrils (Figure 3B, pH 2.0). However, additional signals between 2.5 and 3 ppm could not be unambiguously assigned due to the high density of resonances in this region. Moreover, the broadening of the ligand signals in the presence of fibrils is due to alteration of the relaxation time, and therefore is evidence of binding [ 53 ]. When the pH of the fibrils formed at pH 2.0 was adjusted to 7.4, the signals from the aromatic protons disappeared, except the proton H-4 and protons from the three methyl groups, as can be seen in the corresponding STD spectrum (Figure 3B, pH 2.0 to 7.4). Doxycycline proton signals were absent in the 1 H STD NMR spectrum of doxycycline in the presence of TTRwt amyloid structures at pH 4.4 (Figure 3B, pH 4.4). Contrary to what occurred with the other aggregated samples prepared at pH 2.0 with 0.1 M NaCl and pH 7.4 at 60 ◦ C, the sample with TTRwt aggregates and fibrils assembled at pH 4.4 was extremely turbid, and the amyloid content seemed to quickly precipitate in the NMR tube during the assay. At pH 7.4, the doxycycline protons involved in the interaction with the heat-induced fibrils were similar to those at pH 2.0 (Figure 3B, pH 7.4). 2.3. Effect of Doxycycline on TTRwt Fibril Disaggregation The effect of doxycycline on fibril disruption was assessed by DLS, TEM, and in some of the aggregation protocols by turbidity. Preformed TTRwt aggregates and fibrils were incubated both in the absence and presence of a 50 × molar excess of doxycycline relative to tetrameric TTRwt. DLS measurements were carried out every day until the apparent size stabilization of the species in solution was achieved. TEM images were acquired prior to doxycycline addition and after treatment with doxycycline when the sizes of the particle populations (assessed by DLS) had stabilized. Doxycycline, at 50 × the molar concentration of TTRwt, did not have an immediate persistent effect on the aggregates/fibrils prepared previously with the different protocols, taking from 11 to 17 days to reach a maximum observable disruptive effect. At pH 2.0, after 11 days of incubation with doxycycline, fibrils showed a maximum length of 130 nm and were 4 to 7 nm in diameter, which was a clear decrease of the fibril length and diameter observed by TEM, indicating fibril disruption (Figure 2A, pH 2.0). Moreover, DLS results also supported this observation, which was visible as an increase in particle populations between 100 and 1000 nm (Figure 2B, pH 2.0). Turbidity assays were also carried out to follow TTRwt amyloid fibril aggregation and disaggregation in the presence of doxycycline. However, no signal in turbidity intensity was observed, both in treated and untreated samples with doxycycline (data not shown). This result may be explained by the formation of a clear and completely transparent solution, thus implicating no light scattering signal detection in the pH 2.0 samples, especially when compared to turbid solutions as is the case of pH 4.4 samples. When the fibrils assembled at pH 2.0 underwent pH adjustment to 7.4 and were then treated with doxycycline, the disruptive effect was visible after 17 days both by TEM, with a decrease in the size of the non-fibrillar structures with diameters of 4 nm (Figure 2A, pH 2.0 to 7.4), and DLS, with the disappearance of particle populations equal to or larger than 1000 nm (Figure 2B, pH 2.0 to 7.4). The disruptive effect of doxycycline was also observed in the pre-assembled aggregates/fibrils at pH 4.4 after 17 days of incubation. A decrease in the size of fibrillar structures following treatment with doxycycline was observed by TEM (maximum of 15 nm in length and diameter between 3 and 4 nm) (Figure 2A, pH 4.4). DLS results showed that the control sample had sizes ranging between 1000 and 10,000 nm, whereas in the doxycycline-treated sample, a decrease in the larger size particle populations and an increase in the smaller size populations between 100 and 1000 nm was noticeable (Figure 2B, pH 4.4). Additionally, a turbidity assay was also performed to further characterize the disruptive effect of doxycycline over time in preformed amyloid material (Figure 4). A more pronounced decrease in the turbidity of doxycycline-treated samples was observed at day 11, demonstrating that the aggregated species in solution were smaller or/and in Int. J. Mol. Sci. 2022,23, 391 8 of 17 smaller amount. This effect increased in the following days, reaching a maximum at day 17 with an aggregation estimate of approximately 50% of the control. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 17 The disruptive effect of doxycycline was also observed in the pre-assembled aggregates/fibrils at pH 4.4 after 17 days of incubation. A decrease in the size of fibrillar structures following treatment with doxycycline was observed by TEM (maximum of 15 nm in length and diameter between 3 and 4 nm) (Figure 2A, pH 4.4). DLS results showed that the control sample had sizes ranging between 1000 and 10,000 nm, whereas in the doxycycline-treated sample, a decrease in the larger size particle populations and an increase in the smaller size populations between 100 and 1000 nm was noticeable (Figure 2B, pH 4.4). Additionally, a turbidity assay was also performed to further characterize the disruptive effect of doxycycline over time in preformed amyloid material (Figure 4). A more pronounced decrease in the turbidity of doxycycline-treated samples was observed at day 11, demonstrating that the aggregated species in solution were smaller or/and in smaller amount. This effect increased in the following days, reaching a maximum at day 17 with an aggregation estimate of approximately 50% of the control. Figure 4. Effect of doxycycline on preformed TTRwt fibrils at pH 4.4 monitored by turbidity. Samples of preformed TTRwt fibrillar species (3.6 μM) were incubated both in the absence (control) and presence of 50× molar excess of doxycycline (180 μM) for the indicated time, at room temperature. When the pre-assembled aggregates and fibrils at pH 4.4 underwent pH adjustment to 7.4 and were treated with doxycycline, the disruptive effect was also observed. After 17 days of incubation with doxycycline, the aggregates were 4 to 5 nm in diameter, as observed by TEM images. Moreover, although the fibrillar structures maintained their approximate diameter (4 nm) and length (maximum of 25 nm), they had a lower frequency of occurrence when compared to the untreated samples (Figure 2A, pH 4.4 to 7.4). The DLS results showed that, at this time, the particle populations of doxycycline-treated samples were comprised of particles with sizes between 100 and 1000 nm, whereas in the control samples the majority of the particle populations were higher than 1000 nm, which supports a fibril disaggregation mechanism (Figure 2B, pH 4.4 to 7.4). The disruptive effect of doxycycline was also observed in the preformed heat-induced TTRwt fibrils at pH 7.4. The TEM images acquired after 17 days of treatment showed fibrillar structures exhibiting diameters between 3 to 4 nm and lengths between 10 to 25 nm, and a change in morphology to more amorphous structures in some cases (Figure 2A, pH 7.4). At this incubation time, the DLS results showed the doxycyclinetreated samples were comprised of particles on average smaller, with sizes between 100 and well below 10,000 nm, whereas in control samples with no doxycycline the major particle populations were above 1000 nm (Figure 2B, pH 7.4). Thus, doxycycline also induces fibril disruption of heat-induced TTRwt fibrils. Turbidity assays were also performed in order to follow TTRwt amyloid fibril disaggregation in the presence of doxycycline. However, the results showed a slight increase in the turbidity intensity, when compared to control samples (data not shown), which might be explained by the large amount of smaller species formed during the disaggregation induced by doxycycline, in particular amorphous species, as mentioned previously, leading to higher light scattering intensity Aggregation (%) Figure 4. Effect of doxycycline on preformed TTRwt fibrils at pH 4.4 monitored by turbidity. Samples of preformed TTRwt fibrillar species (3.6 µ M) were incubated both in the absence (control) and presence of 50×molar excess of doxycycline (180 µM) for the indicated time, at room temperature. When the pre-assembled aggregates and fibrils at pH 4.4 underwent pH adjustment to 7.4 and were treated with doxycycline, the disruptive effect was also observed. After 17 days of incubation with doxycycline, the aggregates were 4 to 5 nm in diameter, as observed by TEM images. Moreover, although the fibrillar structures maintained their approximate diameter (4 nm) and length (maximum of 25 nm), they had a lower frequency of occurrence when compared to the untreated samples (Figure 2A, pH 4.4 to 7.4). The DLS results showed that, at this time, the particle populations of doxycycline-treated samples were comprised of particles with sizes between 100 and 1000 nm, whereas in the control samples the majority of the particle populations were higher than 1000 nm, which supports a fibril disaggregation mechanism (Figure 2B, pH 4.4 to 7.4). The disruptive effect of doxycycline was also observed in the preformed heat-induced TTRwt fibrils at pH 7.4. The TEM images acquired after 17 days of treatment showed fibrillar structures exhibiting diameters between 3 to 4 nm and lengths between 10 to 25 nm, and a change in morphology to more amorphous structures in some cases (Figure 2A, pH 7.4). At this incubation time, the DLS results showed the doxycycline-treated samples were comprised of particles on average smaller, with sizes between 100 and well below 10,000 nm, whereas in control samples with no doxycycline the major particle populations were above 1000 nm (Figure 2B, pH 7.4). Thus, doxycycline also induces fibril disruption of heat-induced TTRwt fibrils. Turbidity assays were also performed in order to follow TTRwt amyloid fibril disaggregation in the presence of doxycycline. However, the results showed a slight increase in the turbidity intensity, when compared to control samples (data not shown), which might be explained by the large amount of smaller species formed during the disaggregation induced by doxycycline, in particular amorphous species, as mentioned previously, leading to higher light scattering intensity when compared to the short and unbranched fibrillar structures observed in the control sample (Figure 2A, pH 7.4). 3. Discussion With the goal of finding an effective and relatively fast screening protocol to search for TTR amyloid fibril disruptors, we have reviewed, characterized and selected the most appropriate TTR aggregation protocol among those previously described. After a careful analysis of the available literature, three TTRwt aggregation protocols were selected for comparison, performed at different pH values: pH 7.4, pH 4.4, and pH 2.0 (Tables 1and 2). The protocol involving heat induction at pH 7.4 was selected since it is performed at the pH of physiological conditions [ 51 ]. Acidification is the most widely used strategy to induce TTR aggregation. At pH 4.4 the TTR tetrameric structure dissociates into monomeric species that undergo partial unfolding and, consequently, associate to form amyloid structures [ 21 ]. Int. J. Mol. Sci. 2022,23, 391 9 of 17 Acidification at pH 2.0 promotes tetramer dissociation followed by denaturation, and the addition of 0.1 M NaCl induces aggregation, since chloride ions shield the positive charges of the protein, allowing for conformational changes and the assembly of monomers into oligomers and amyloid fibrils [ 54 ]. Thus, aggregation processes are extremely dependent not only on pH changes, but also on protein concentration, ionic strength, temperature, and agitation. The aggregation protocol of TTRwt at pH 2.0 with 0.1 M NaCl has been previously well characterized and its kinetics studied by our research group [ 20 ]. This is the most time-consuming of the three protocols, and it requires a relatively high concentration of tetrameric TTRwt to be effective in an acceptable time period. Aggregation at this pH, with a physiological plasma protein concentration (3.6 µ M), was also performed and the process was followed by DLS for approximately 30 days. However, no significant changes in particle populations were observed during the experiment (data not shown), which indicates that DLS could not detect fibril formation in this period of time at 3.6 µ M TTR. The fibrils formed at pH 2.0 are unbranched, long, well-formed and mature (Figure 2A, pH 2.0), and bind ThT (Table 2), a good indication of presence of amyloid structures. Adjustment of pH to 7.4 of TTRwt fibrils preformed at pH 2.0 was an attempt to mimic the in vivo conditions of TTRwt fibrils and to be able to study the action and efficacy of potential disruptors in close to physiological conditions. TTRwt amyloid deposits are mostly extracellular [ 55 ] where pH is generally close to neutral. The effect of pH modification on the size, structure, and morphology of preformed TTRwt amyloid was studied by DLS, CD, and TEM experiments, respectively. The DLS results showed that incubation in phosphate buffered saline (PBS), at pH 7.4, of TTRwt amyloid fibrils preformed at pH 2.0 led to a decrease in the average size of the particles, with populations of particles smaller than 1000 nm being more common (Figure S1B). The CD results revealed a loss of β -sheet secondary structure (Figure 1A and Table 3), and the TEM images corroborated the previous observations showing that fibrils lose their morphology, appearing as smaller fibrils and amorphous aggregates (Figure 2A, pH 2.0 to pH 7.4); however, they still remained ThT positive (Table 2). The fibril formation protocol at pH 4.4 is widely used for in vitro TTRwt amyloid fibrillogenesis inhibitor screening, as it is easily implemented in medium or high throughput screening formats in tubes or microwell plates, usually followed by turbidity measurements [ 56 – 58 ]. Thus, this protocol seems to be a very good candidate for use in TTRwt disaggregation assays. This method is less time-consuming than the previous method at pH 2.0, does not require high concentrations of native TTRwt, and amyloid species can be formed at physiological concentrations (3.6 µ M) after only 3 days (Figure S4). The formation of amyloid aggregates and fibrils using this protocol can be monitored by both DLS and turbidity. In an attempt to follow aggregation, CD was also performed every day for 3 days; however, the spectra remained unaltered and similar to Figure 1B. At pH 4.4, the species formed are composed by small fibrils and aggregates, while samples remained ThT positive (Table 2). pH adjustment to 7.4 was also performed for these amyloid structures, with CD (Figure 1B), DLS (Figure 2B, pH 4.4 to pH 7.4), and TEM (Figure 2A, pH 4.4 to pH 7.4) assays demonstrating that the pH change slightly altered the structure, size, and morphology of these aggregates; however, they remained ThT positive (Table 2). The third aggregation protocol is the least studied process of the three. Heat-induced fibril formation at pH 7.4 seems to be a very good candidate for use in TTRwt fibril disruptor screening assays because avoids the influence of low pH on the mechanism of action of the disruptor. 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