Characterization of conformation-specific antibodies TOMA-1 and TTCM-1 for recombinant tau monomer and amplified brain derived tau oligomer

Abstract

Tauopathies are a set of neurodegenerative diseases characterized by the pathological accumulation of aggregated tau in the brain. Recent breakthrough evidence has revealed the existence of different strains of tau oligomer (TauO) which direct the different pathological presentation of individual tauopathies. Extensive research efforts have been devoted to search for specific antibodies or drug candidates which target TauO to serve as promising alternatives to treat Alzheimer’s disease (AD) in future. To screen for the antibodies which are able to bind with amplified brain derived tau oligomer (aBDTO), we have investigated the binding parameters of the tau oligomer-specific monoclonal antibody-1 (TOMA-1) and toxic tau conformation-specific monoclonal antibody-1 (TTCM-1) with the recombinant tau monomer (rTauM) and aBDTO using isothermal titration calorimetry (ITC). TOMA-1 specifically recognizes the amino acid sequences 367–386, 382–401 and 367–386 and TTCM-1 specifically recognizes the amino acid sequence 307–326 of rTauM and aBDTO, respectively. Our results demonstrated that both TOMA-1 and TTCM-1 have a high binding affinity with aBDTO compared to rTauM. We also observed that higher the binding affinity of the antibody to the aBDTO, lower was the toxicity of the aBDTO and vice versa. Our study taken together presents both TOMA-1 and TTCM-1 to be potential immunotherapeutic agents against AD.

1. Introduction

Pathologically, neurodegenerative diseases share the common features associated with the accumulation of aggregated proteins in the brain. The protein aggregation process is initiated by the misfolded monomers which further assemble into high molecular weight (HMW) tau forms that become more resistant to degradation [1–3]. The most commonly aggregating proteins in AD are phosphorylated tau (presenting itself as neurofibrillary tangles) and extra cellular beta-amyloid (Aβ) which forms Aβ-plaques [4–7]. Complex pattern of post translational modifications dynamically regulates tau function by regulating its biochemical properties [8]. Tauopathies such as AD, chronic traumatic encephalopathy, corticobasal degeneration (CBD) and Pic’s disease (PiD) are caused due to the predominant accumulation of misfolded tau [9]. Mature fibrillar species of amyloidogenic proteins were once considered to be the most toxic forms, but now recent research has shifted their focus on smaller, soluble aggregates, often referred to as oligomers [10–12]. Enhanced toxicity of tau oligomer (TauO) is due to its high solubility which contributes to its higher reactivity, cellular interaction, seeding behavior and their consequent easy propagation [13–15]. These findings have shifted the focus of therapeutic strategies to target oligomeric species to mitigate the toxic effect of oligomers and potentially halt or reverse the progression of the associated diseases [16–24]. The conformation of tau aggregates can be identical within the same disease, but it often varies across different tauopathies. Due to this variability of tau aggregate conformation across different tauopathies, the term tau strain has originated [9].The investigations on different tau strains are ongoing and it mainly focusses on understanding the mechanisms underlying their formation and their implication in disease progression [9,25]. The spread of tau pathology throughout the brain is mediated by tau seeds which are the misfolded tau protein acting as a template for misfolding and aggregation of the normal tau. The tau seeds are composed of either 3R (having R1, R3 and R4 and either 0 N, 1 N, or 2 N repeats) or 4R (having R1, R2, R3 and R4 and either 0 N, 1 N, or 2 N repeats) and are spread via synapse [5,26,27]. The microtubule binding region (MTBR) of tau plays a critical role in the formation of different neuropathological aggregates identified in AD brain and other tauopathies. Various approaches are being considered to slow down or stop the progression of tau pathology, for example, the use of small molecules [28–30] and immunotherapeutic approaches using anti-tau antibodies [31–34]. Targeting the MTBR with a therapeutic antibody to reduce the seeding potential of tau may be a promising step towards the development of immunotherapeutic against AD and other tauopathies [27]. Currently, the lack of information on the structure of tau strains limits our understanding of the progression of neurodegeneration. To elucidate the role of the tau oligomer-specific monoclonal antibody-1 (TOMA-1) and toxic tau conformation-specific monoclonal antibody-1 (TTCM-1) in the treatment and management of AD and other tauopathies, we have measured the binding parameters of the two isoforms of tau (2N3R and 2N4R) in monomeric (rTauM) and oligomeric (aBDTO) forms with both the antibodies. As their name suggested, TOMA-1 recognizes tau oligomers and TTCM-1 recognizes toxic form of tau (tau oligomers and protofibrils). We determined the dissociation constant (K_d), stoichiometry (n), enthalpy (ΔH), entropy (ΔS) and change in Gibbs free energy (ΔG) of the rTauM and aBDTO with TOMA-1 and TTCM-1. The epitopes for the TOMA-1 and TTCM-1 clones that have been mapped using the methods developed by Reyes-Ruiz et al. [35,36], recognize non-continuous epitopes within the MTBR region. From the studies on the SH-SY5Y cells treated with aBDTO together with TOMA-1 and TTCM-1, it was observed that there was a reduction in the toxicity and seeding potential of the aBDTO. This may be due to the binding of TOMA-1 and TTCM-1 at MTBR region which somehow modulates the structure or conformation of the tau aggregates, because of the formation of immune-complexes (protein-antibody complexes). All the results taken together provide novel insights into using conformation-specific antibodies for modulating the aBDTO toxicity. Both TOMA-1 and TTCM-1 represent themselves as powerful immunotherapeutic tools for the treatment and management of AD [37].

2. Results

2.1. Amplification of BDTO using rTauM

To obtain the aBDTO of Tau2N3R and Tau2N4R, we recombinantly expressed the monomeric forms of these proteins in Escherichia coli BL21. Next, the expressed proteins were purified using affinity chromatography (Ni-NTA), we isolated the tau monomers from the purified protein and determined their purity by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (Fig. S1A). To start the amplification of BDTO, un-aggregated rTauM was rotated at 10–15 rpm at 37 °C for 48 h in the presence of BDTO (seed). The ratio of rTauM and BDTO used for the amplification reaction was 100:1. This ratio was used to minimize the effect of seed on the results [38]. The rTauM and BDTO mixture was subjected to continuous rotation at 15–20 rpm for 48 h at 37 °C. The aBDTO thus formed was characterized using SDS-PAGE, size exclusion chromatography (SEC) for quality control (Fig. S1).

2.2. Structural characterization of rTauM and aBDTO by far-UV circular dichroism (CD) and fluorescence

To see the changes in the secondary structure of the two isoforms of rTauM and aBDTO, we used far-UV CD in the wavelength range of 195 nm–250 nm (Fig. 1A–B). A prominent negative peak at 200 nm for rTauM (2N3R and 2N4R) is indicative of the presence of random coil conformation. The far UV-CD spectra of aBDTO (2N3R and 2N4R) show a peak shift towards higher wavelength, indicating the formation of β-sheet structure in aBDTO (of both 2N3R and 2N4R). Further, the aggregation state of aBDTO (2N3R and 2N4R) were monitored by ThT fluorescence at 490 nm upon excitation at 450 nm. ThT fluoresced strongly upon binding to the cross β-sheets and allowed us to perform qualitative and quantitative analysis of amyloid content of aBDTO (Fig. 1 C–D).

Fig. 1.

Characterization of rTauM and aBDTO) using (A) Far UV-CD of Tau2N4R (monomer and aBDTO), (B) Far UV-CD of Tau2N3R (monomer and aBDTO). Dye Biding Assay using ThT (C) 2N3R incubated with TOMA-1 and TTCM-1 (D) 2N4R incubated with TOMA-1 and TTCM-1.

2.3. Morphological features of aBDTO and aBDTO-antibody complex

The morphology of aBDTO alone and its complex with the antibody (TOMA-1 and TTCM-1) were further investigated using atomic force microscopy (AFM) (Fig. S2). AFM images of different samples are displayed in Fig. S2 A–F, whereas height and diameter distribution after particle analysis are depicted on the right side of respective images. In the sample having aBDTO alone (Fig. S2 A and D) different sizes of the oligomers were formed (both small and large) in terms of height and diameter. While the aBDTO incubated with antibody was found to lack large size oligomers (height > 2 nm and diameter > 20 nm) (Fig. S2 B, C, E and F).

2.4. Determination of hydrodynamic radii of rTauM and aBDTO alone and in complex with antibody

The size distributions were evaluated for rTauM, aBDTO, aBD-TO+TOMA-1, aBDTO+TTCM-1 (for both 2N3R and 2N4R tau isoforms), TOMA-1 and TTCM-1. The intensity versus diameter was plotted in logarithmic scale (Fig. 2). Fig. 2A represents the comparisons of size distribution for rTauM, aBDTO, aBDTO+TOMA-1 and aBDTO+TTCM-1 (2N4R isoform). The average diameter of rTauM, aBDTO, antibody (TOMA-1 and TTCM-1) were found to be 9 nm, 12 nm and 10 nm, respectively. Complexes of TOMA-1 and TTCM-1 with aBDTO have large size (15–30 nm) which manifests the binding and formation of aBDTO-antibody complexes. The other peaks observed in the samples around 2 nm, indicate the degradation of some monomers and aBDTO (Fig. 2A). In Fig. 2B, the size distribution of rTauM, aBDTO, aBDTO+TOMA-1 and aBDTO+TTCM-1 (2N3R isoform) is compared. Additional peaks between 20 and 30 nm represent the formation of larger aggregates in aBDTO and aBDTO+TTCM-1 whereas small peak at <5 nm signifies degradation of rTauM (Fig. 2B).

Fig. 2.

Homogeneity of samples were evaluated by size distribution versus intensity of rTauM, aBDTO (A) 2N4R alone, aBDTO incubated with TOMA-1 or TTCM-1 (1:10 ratio) and antibody alone; (B) 2N3R alone, aBDTO incubated with TOMA-1 or TTCM-1 (1:2 ratio) and antibody alone. Samples were filtered and centrifuged to remove any dust or aggregated forms. Measurements were done at fixed angle (90°) and constant temperature at 20 °C.

2.5. Binding affinity of antibodies with rTauM and aBDTO

ITC was used to evaluate the interaction between conformation-specific antibodies and the proteins (rTauM and aBDTO) (Figs. 3 and 4). The three-dimensional structure and flexibility of complementarity determining regions (CDRs) of an antibody determines its specificity, selectivity and binding strength with the epitopes on amyloid polymorphs [39–42]. Conformation-dependent monoclonal antibodies against oligomeric, fibrillar forms of Aβ and amyloid precursor proteins are extensively studied [36,42,43]. Excitingly, now we have mapped the epitopes of the TOMA-1 and TTCM-1 clones using the methods developed by Reyes-Ruiz et al. [35,36] for mapping epitopes for conformation-specific anti amyloid antibodies. These novel TOMA-1 and TTCM-1 clones recognize the non-continuous epitopes within the MTBR region far away from the N-terminal disordered region of tau. We found that the monoclonal antibodies recognize the specific sequence of rTauM and aBDTO for both the isoforms. TOMA-1 binds at sequences 367–386, 382–401, and 367–386 and TTCM-1 binds at 307–326 in the MTBR of tau, which is the key region for PHF (paired helical filaments) formation. We hypothesize that the toxic tau conformation-specific antibodies, TOMA-1 and TTCM-1 could clear toxic TauO from extracellular space or prevent them from seeding further [31]. We examined the various binding parameters such as dissociation constant (K_d), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) of conformation-specific antibodies (TOMA-1 and TTCM-1) with rTauM and aBDTO. Dissociation constant (K_d) of antibody-protein complex is used as a determinant of specificity and selectivity, which is important for the safety and efficacy of the anti-tau therapy. The K_d values for the TOMA-1 and TTCM-1 with respect to rTauM and aBDTO indicate that aBDTO exhibits a stronger interaction with antibodies compared to rTauM (Table 1).

Fig. 3.

ITC endotherm of 2N3R isoform, rTauM with (A) TOMA-1 (B) TTCM-1, and aBDTO with (C) TOMA-1 (D) TTCM-1 at pH 7.4 and 25.0 ± 0.1 °C. Upper panel of each figure shows raw data representing each injection of ligand. The peaks were normalized to the protein/ligand molar ratio and were integrated as shown in lower panel. Inset in the figure shows the signature plot.

Fig. 4.

ITC endotherm of 2N4R isoform, rTauM with (A) TOMA-1 (B) TTCM-1 and aBDTO with (C) TOMA-1 (D) TTCM-1 at pH 7.4 and 25.0 ± 0.1 °C. Upper panel of each figure shows raw data representing each injection of ligand. The peaks were normalized to the protein/ligand molar ratio and were integrated as shown in lower panel. Inset in the figure shows signature plot.

Table 1.

Thermodynamic parameters associated with the binding of TTCM-1 and TOMA-1 with rTauM and aBDTO.

Tau-isoform	Antibody	rTauM/aBDTO	Stoichiometry (n)	Kd μM	ΔH kJmol−1	-TΔS kJmol−1	ΔG kJ mol−1
Tau	TOMA-1	rTauM	2.01 ± 0.29	5.02 ± 0.07	−331.0 ± 21.2	300.7	−30.3
2N3R		aBDTO	1.10 ± 0.14	0.91 ± 0.49	−334.7 ± 34.7	306.3	−27.9
	TTCM-1	rTauM	1.62 ± 0.13	1.15 ± 0.08	−261.3 ± 30.5	228.4	−33.9
		aBDTO	1.15 ± 0.19	0.19 ± 0.02	−53.6 ± 1.3	38.28	−15.1
Tau	TOMA-1	rTauM	1.49 ± 0.09	1.04 ± 0.10	−290.8 ± 24.7	256.1	−34.9
2N4R		aBDTO	0.52 ± 0.08	0.23 ± 0.03	−334.7 ± 25.5	296.6	−38.2
	TTCM-1	rTauM	1.70 ± 0.22	3.22 ± 0.90	−334.7 ± 33.9	303.3	−31.4
		aBDTO	0.52 ± 0.07	0.20 ± 0.05	−336.0 ± 30.1	295.0	−41.0

From the Table 1 it is evident that the binding interaction was enthalpy driven. The favourable free energy (ΔG) of binding is majorly contributed by enthalpy which suggests that the binding is favored by exothermic and stabilizing interactions. Furthermore, the interaction involves hydrogen bonds which indicate that the interaction is stabilized by non-covalent forces adding to the overall strength and specificity. The stoichiometry of binding indicates the nature of antibody-tau complex which depends on the aggregation state of tau. The stoichiometry of TTCM-1 and TOMA-1 for the 2N4R rTauM ranges from 1.5 to 1.7 (Table 1), indicating that single antibody binds to two tau epitopes. Whereas the stoichiometry of antibodies for aBDTO is 0.5, which implies that single aBDTO binds with both the variable regions of the antibody. However, in case of 2N3R isoform, the stoichiometry of TTCM-1 and TOMA-1 with aBDTO is changed from 0.5 to 1 which implies overlapping binding epitopes (Table 1 and Inset of Figs. 3 and 4).

Distinct selectivity was evaluated by binding assay of two different antibodies against different conformations of tau (rTauM and aBDTOs for 2N4R (Fig. 5A) and 2N3R (Fig. 5B)). It can be observed that TTCM-1 binds more strongly and more specifically (demonstrated by color gradient) to the 2N4R aBDTO. The color gradient from left to right indicates the increasing K_d values) (Fig. 6).

Fig. 5.

Binding parameter of TOMA-1 and TTCM-1 against different isoforms of rTauM and aBDTO (A) 2N4R and (B) 2N3R. The Figure indicates that TTCM-1 binds strongly (dark grey color) to aBDTO and is more specific to the 2N4R isoforms (Color gradient from left to right indicates increasing K_d values).

Fig. 6.

aBDTO exhibits cytotoxicity in SH-SY5Y cell line and neutralize by TOMA-1 and TTCM-1 (A) aBDTO was pre-incubated with IgG as control, TOMA-1 and TTCM-1 at the 1:2 ratio of aBDTO+antibody for 1 h at room temperature and added to the SH-SY5Y cells for 24 h; Cytotoxicity was analyzed by LDH assay. Data were compared by ordinary one-way ANOVA followed by Turkey’s multiple comparisons test where *indicates values compared with UT and # indicated values compared with aBDTO, *p < 0.05; ****p < 0.0001. (B) Representative of immunofluorescent images of h-Tau primary neurons treated with aBDTOs or aBDTO+antibody mixture. Neurons were fixed and immunolabeled with total tau (Tau 13, magenta), phosphorylated tau pSer422 (red), neuronal marker (NeuN, green), and mature neuron (βIII-tubulin, blue). (C) Analysis of the cellular tau intensity showed a significant increase of tau intensity in aBDTO incubated with TTCM-1 antibody compared to aBDTO incubated with TOMA-1 and aBDTO alone treated group. Ordinary one-way ANOVA AND Dunnett’s multiple comparisons test, significance p < 0.05, ** is reported. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.6. Cytotoxicity assay and immunostaining

The cytotoxic effects of rTauM and aBDTO were analyzed in the presence and absence of tau conformation-specific antibodies (TOMA-1 and TTCM-1). The confluent culture of human neuroblastoma SH-SY5Y cells was treated with 2.0 μM aBDTO alone and 2.0 μM aBDTO with 4 μM antibody (aBDTO+antibody) in 1:2 ratio. The treated cells were then incubated for 24 h. The cell viability for the aBDTO and aBDTO+antibody treated cells was compared. It was observed that the cells treated with aBDTO alone were less viable compared to the ones treated with aBDTO+antibody in 1:2 ratio. This implies that the cytotoxicity of the aBDTO is reduced in the presence of the antibodies, TTCM-1 and TOMA-1. The toxicity of aBDTO was 0 % in TTCM-1 treated cells and was reduced to ~50 % in TOMA-1 treated cells (Fig. 6A). The immunofluorescence images of h-Tau primary neurons treated with aBDTO alone and aBDTO+antibody in 1:2 ratio is shown in Fig. 6B and C. The neurons were fixed and immunolabeled with total tau (Tau 13, magenta), phosphorylated tau pSer422 (red), neuronal marker (NeuN, green), and mature neuron (βIII-tubulin, blue). Increased tau intensity was observed in cells treated with aBDTOTTCM-1 in 1:2 ratio, compared to cells treated with aBDTO-TOMA-1 (1:2) or aBDTO alone. This result indicates that TTCM-1 and TOMA-1 modulate aBDTO to assume non-toxic form which is detected by Tau 13 and tau pSer422. This result correlates well with the reduced toxicity of aBDTO in the presence of antibodies.

3. Discussion

Diverse forms of tau exist in nature as monomers, oligomers, protofibrils and fibrils with a plethora of physiological roles. Among all, oligomers and protofibrils are the most toxic forms of tau and further aggregation and accumulation of these toxic forms in the brain is a common feature of various tauopathies [44,45]. Reduction of these toxic tau aggregates in neuronal cells have shown to mitigate the Aβ-mediated toxicity [46]. Recently, researchers clearly demonstrated that the toxicity of soluble tau oligomers is due to their seeding behavior and the ability to propagate through the tissues because of their high solubility [13–15]. For developing therapeutic strategies against toxic tau aggregates, especially targeting tau oligomers, using small molecules [28] and tau specific antibodies [31,47,48] are becoming more promising. Antibody mediated therapies are intended to ameliorate or prevent the buildup of toxic oligomer of Aβ [49–53], α-synuclein [54] and tau [31,55,56]. For this study, we utilized rTauM expressed in bacterial system for this study and generation of aBDTO. For characterization of rTauM and aBDTO, we use SDS-PAGE and size exclusion chromatography for purity (Fig. S1 A–B) and far-UV CD to see secondary structure difference between rTauM and aBDTO (Fig. 1 A–B). Conformation changes in aBDTO and aBDTO-antibody complex were further investigated by florescence probe (Fig. 1 C–D). The reduced emission intensity and to some extent the red shift in wavelength maxima indicated loss of β-sheet rich aggregated conformation in the presence of antibody. Analysis of morphological features using AFM results of aBDTO alone (Fig. S2 A and D) and aBDTO incubated with antibody (Fig. S2 B, C, E and F) are similar except that large dimension oligomers disappeared in the presence of conformation specific antibody. Size distributions of rTauM, aBDTO and aBDTO-antibody complex were examined in Fig. 2 providing additional information about the heterogeneity and dynamics of aBDTO in solution.

Our final goal is to estimate the binding parameters associated with TOMA-1 and TTCM-1, with rTauM and aBDTO of both 3-repeat (3R) and 4-repeat (4R) tau isoforms using ITC (Figs. 3 and 4). Conformation specific antibodies, TTCM-1 and TOMA-1 were previously generated in our laboratory [57]. Antibodies (TOMA-1 and TTCM-1) are designed to specifically recognize and bind with tau oligomer and toxic tau conformation hence they derive their name; tau oligomer-specific monoclonal antibody (TOMA-1) and toxic tau conformational monoclonal antibody (TTCM-1), respectively [57]. The binding of the antibodies with rTauM is weak as indicated by the K_d value of 5.02 μM and 3.22 μM for TOMA-1 and TTCM-1, respectively, as demonstrated by ITC results. TOMA-1 and TTCM-1 both showed relatively higher binding to the aBDTO compared to their respective tau monomers, with a 5-fold and 15-fold higher affinity with 2N4R and 2N3R, respectively (Table 1). These results demonstrate that both antibodies have high selectivity to bind with the aBDTO as compared to rTauM. DLS measurement of rTauM and aBDTO in the absence and presence of TOMA-1 and TTCM-1 confirmed the formation of tau-antibody complexes which further supports our ITC results (Fig. 2).

We have also investigated whether the toxicity of aBDTO-antibody mixture was less compared to aBDTO alone in SH-SY5Y cells using LDH assay. SH-SY5Y cells treated with aBDTO-antibody complex showed reduced toxicity. The cells treated with aBDTO+TOMA-1 and aBDTO+TTCM-1 displayed 50 % and 0 % toxicity, respectively, compared to the cells treated with aBDTO alone (Fig. 6A). Immunostaining of h-Tau primary neurons treated with aBDTO+TTCM-1 displayed increased intensity for Tau 13 and tau pSer422, compared to aBDTO treated cells (Fig. 6; B and C). All these results, taken together, support that both TTCM-1 and TOMA-1 are highly specific for aBDTO and the toxicity of the aBDTO is reduced in the presence of the both the antibodies.

4. Conclusion

Extensive research efforts have been devoted to search for specific antibodies that could specifically target the toxic oligomers for the development of immunotherapeutic against AD. ITC binding study showed that TOMA-1 and TTCM-1 demonstrate a pronounced binding preference for the aBDTO compared to rTauM for both the isoforms of tau protein. Further, both these antibodies displayed higher affinity for the aBDTO of 2N4R isoform compared to 2N3R aBDTO. Our finding suggests that both TOMA-1 and TTCM-1 modulate the tau aggregates by forming immune-complexes. This leads to reduced aBDTO toxicity and seeding potential, which is well supported by the investigations on the SH-SY5Y cells and h-Tau primary cortical neurons. Taken together, these antibodies present themselves as a powerful immunotherapeutic tool to treat and manage AD and other tauopathies.

5. Material and methods

5.1. Expression and purification of recombinant tau

Expression and purification of recombinant tau (2N4R and 2N3R) monomer was performed as previously described [58–60]. Briefly stating, we used E. coli BL21 (DE3) competent cells (New England Biolabs) and transformed them with plasmid (pET29b) harboring MAPT gene, to express full length tau protein (441amino acid). The glycerol stocks of the transformed BL21 (DE3) cells were revived as primary culture of 20 mL Luria-Bertani (LB) broth, supplemented with 0.1 mg/mL kanamycin. The primary culture was allowed to grow for 16 h at room temperature (RT), with shaking at 200 rpm. Following this, secondary culture was prepared by inoculating 1 L of LB broth containing 0.1 mg/mL kanamycin with 20 mL of the primary culture. The secondary culture was allowed to grow at 37 °C with shaking at 200 rpm on orbital shaker incubator till the value of the O⋅D₆₀₀ (Optical density cell culture at 600 nm) reached 0.6–0.8. This was followed by induction of protein expression in the secondary culture with 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) and allowing the cells to grow at 25 °C, with shaking at 200 rpm for ~16 h. Cells were then harvested by centrifugation at 5000 rpm for 10 min at 4 °C. The cell pellets thus obtained were re-suspended in 25 mL lysis buffer (20 mM Tris, 500 mM NaCl and pH 8.0) supplemented with one cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) and 1 mM PMSF. The cells were then lysed by sonication using 30s on/off cycle at 50 % amplitude for 20 min on ice. The cell lysate was centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was collected and boiled at 90 °C for 30 min to precipitate the protein impurities and contaminants. After boiling, the supernatant was centrifuged at 12000 rpm for 15 min at 4 °C. After centrifugation, the supernatant was collected and incubated with pre-equilibrated HisPur™ Ni-NTA resin (Thermo Scientific) for a minimum of 30 min at 4 °C with continuous shaking. The mixture was then packed into a Kontes^® flex-column. The column was washed with 2 bed volumes of wash buffer 1 (20 mM Tris, 500 mM NaCl, and pH 8.0), 1 bed volume of wash buffer 2 (20 mM Tris, 500 mM NaCl, 10 mM imidazole and pH 8.0) and the protein was eluted in 1 bed volume of elution buffer (20 mM Tris, 500 mM NaCl, 100 mM imidazole and pH 8.0). Following this, we exchanged the elution buffer for 1×PBS, using Slide-A-Lyzer™ Dialysis Cassettes of 20 kDa MWCO (Thermo Scientific). Size exclusion chromatography (SEC) was used for further purification of tau monomer. 5 mL protein sample was loaded on to HiLoad 16/600 Superdex 75 pg column (Cytiva, USA) preequilibrated in phosphate buffer with flow rate of 1 mL/min. SDS-PAGE followed by silver staining was performed after each purification step to monitor the purity of the protein at each step (Fig. S1A). The concentration of the protein was estimated using BCA assay and the protein was suitably aliquoted and snap-frozen for storage at −80 °C until further use.

5.2. Amplification of BDTO

The isolation and purification of BDTO from the human AD brain tissue and the amplification of this BDTO by seeding of rTauM (2N4R and 2N3R) was performed as previously described [61]. To amplify the BDTO, 20 μM rTauM and 0.2 μM BDTO (in 100:1 ratio) in 1× PBS (Gibco) was incubated at 37 °C for 48 h while rotating the mixtures at 15–20 rpm. The amplification mixtures were prepared in low-retention micro-centrifuge tubes (Fisher brand). Amplified BDTO was confirmed by small scale SEC to visualize aBDTO and rTauM was used as a control. 20 μg of protein was loaded on to Superdex 200 Increase 10/300gl column, with flow rate of 0.75 mL/min) (Fig. S1B).

5.3. Circular dichroism (CD)

Far-UV CD spectra of rTauM (2N4R and 2N3R) and aBDTO were scanned using Jasco J-815 CD spectropolarimeter equipped with Peltier type temperature controller. For this, we used 0.1 mg/mL protein sample in a quartz cuvette with 1 mm path length. The measurement parameters were set at scan rate of 20 nm/min and an interval of 1 nm to record data from 195 to 250 nm with an average of three iterations was used for each protein (monomers/oligomers) sample. The CD data were extracted using Spectra Manager provided by Jasco (Jasco Inc. Japan). The experiment was performed at least in triplicate and data was plotted using GraphPad Prism and analyzed for secondary structure content using K2D2 algorithm [62–64].

5.4. ThT fluorescence

Fluorescence assays were conducted on a POLARstar Omega plate reader (BMG Lab Technology) in triplicate [65]. 50 μL of each sample was added to a clear bottom 96-well plate ensuring final concentration of protein in the sample remains 2 μM when mixed with 10 μM ThT dye. The samples were incubated at room temperature for 3 h. The ThT emission spectra were measured at an emission wavelength of 482 nm upon excitation at 450 nm. Background corrections were performed by subtraction of baseline fluorescence signal of the buffer and dye alone from corresponding samples. Each sample was tested in triplicate and data were processed using GraphPad Prism.

5.5. Atomic force microscopy (AFM)

AFM was used to characterize aBDTO as described previously [44,66,67]. AFM images of aBDTO were obtained using a non-contacting tapping method with Bruker Multimode 8 AFM. 12 mm mica fixed on a 15 mm metallic disc was freshly cleaved to obtain a uniform surface. 10 μL of 1:10 dilution sample (0.03–0.05 μg/μL) in PBS was applied on mica and allowed to adsorb overnight (~16 h at RT). The mica grid was then repeatedly washed with deionized water and dried in the air. Images were taken at different fields on the mica surface. Finally, images were analyzed using NanoScope Analysis v1.20rl AFM data processing software and data was plotted using GraphPad Prism.

5.6. Dynamic light scattering (DLS)

Size distribution of rTauM (2N4R and 2N3R), aBDTO and complex of protein and antibody under investigation were estimated by DLS (Zetasizer Malvern) using fixed angle of 90° at 20 ± 0.1 °C. Samples were either filtered or centrifuged before experiments to remove dust particles. The concentration of proteins used for the DLS measurements was 1 mg/mL. For each sample, 3 runs of 10 measurements were made for 20s acquisition time. Data was analyzed using software provided by Malvern Inc.

5.7. Isothermal titration calorimetry (ITC)

ITC experiments were conducted using a MicroCal PEAQ-ITC Malvern. The antibodies (TTCM-1 and TOMA-1), rTauM (2N4R and 2N3R) and aBDTO were dialyzed against 1× PBS to circumvent buffer mismatch during the experiments. 2 μM of antibody (TOMA-1/TTCM-1) was placed in the sample cell. The syringe was loaded with 20 μM of tau (rTauM/aBDTO). The rTauM/aBDTO was then titrated into the cell from the syringe in stepwise manner. Each time 3 μL of rTauM/aBDTO was injected in the cell with a time delay of 180 s. For an experiment, a series of 10–15 injections were set at 60s initial delay with 750 rpm stirring speed and at 25 °C. Data was analyzed using Malvern analysis module provided with ITC instrument. Nonlinear regression analysis was performed in a sequential binding model with two binding sites with varying stoichiometry (n), dissociation constant (K_d) and molar binding enthalpy (ΔH) to determine the thermodynamic parameters of binding.

5.8. Cytotoxicity

Cytotoxicity of aBDTO was measured on cultured human neuroblastoma SH-SY5Y cells using LDH release assay (Cytotoxicity Detection Kit^PLUS LDH, Ref-04744934001, Roche) following the manufacturer’s instructions and as previously described [67,68]. Briefly, SH-SY5Y cells were cultured at a density of 10,000 cells/well in 96-well plate at 5 % CO₂ and 37 °C. Cells were treated with 2.0 μM aBDTO alone and aBDTO-antibody in 1:2 ratio, incubated for 24 h and assessed by LDH release assay. Similarly, the primary neuron cells were treated and the cytotoxicity of aBDTO and aBDTO-antibody in 1:2 ratio and assed using immunolabeling. Optical density was measured at 490 nm with POLARstar OMEGA plate reader (BMG Lab technologies). All measurements were performed in triplicate and background was corrected by the blank.

5.9. Primary neuron preparation

h-Tau primary neuronal cultures were prepared and maintained as described previously [15,28]. Briefly, cortical neurons were isolated from human tau expressing mice (Jackson Laboratory; 005491) during embryonic 13–16 days using Accutase solution (Sigma; A6964) together with gentle trituration by a fire-polished glass Pasteur pipette. Dissociated cells were plated on poly-L-lysine coated-coverslips at a density of 2 × 10⁵ cells/mL in a 24-well plate containing neurobasal medium (Gibco; 12,348,017) plus 2 % B-27 Plus, 0.5 mM GlutaMax (Gibco; 35,050–061), 10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B supplement. In all, 50 % of medium changes were performed every 3–5 days. Cells on day 10 in vitro (DIV) were used for all experiments.

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbadis.2025.167995.

Data availability

Data will be made available on request.