The antidepressant drug; trazodone inhibits Tau amyloidogenesis: Prospects for prophylaxis and treatment of AD

Archives of Biochemistry and Biophysics 679 (2020) 108218

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The antidepressant drug; trazodone inhibits Tau amyloidogenesis: Prospects for prophylaxis and treatment of AD

T

Vali Akbaria, Sirous Ghobadia,∗∗, Soheila Mohammadib, Reza Khodarahmic,d,∗ a

Department of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran Nano Drug Delivery Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran c Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran d Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tau prtein Trazodone SH-SY5Y Tau aggregation inhibitor Docking/molecular dynamics Alzheimer's disease

Tau protein, characterized as “natively unfolded”, is involved in microtubule assembly/stabilization in physiological conditions. Under pathological conditions, Tau dysfunction leads to its accumulation of insoluble toxic amyloid aggregates and thought to be involved in the degeneration and neuronal death associated with neurodegenerative diseases. Trazodone (TRZ), a triazolopyridine derivative, is a selective serotonin reuptake inhibitor (SSRI) which increases serotonin levels in synaptic cleft and potentiating serotonin activity, with antidepressant and sedative properties. This drug is more effective and tolerable than other therapeutic agents. In this study, the 1N4R isoform of Tau protein was purified and the effect of TRZ on the protein fibrillation was investigated using multi-spectroscopic techniques as well as computational methods. The results showed that TRZ is not only able to affect formation of Tau amyloid fibrils in vitro but also attenuates Tau oligomerization within SH-SY5Y cell line resulting in more cells surviving. Moreover, membrane disrupting activity of Tau aggregates decreased upon TRZ treatment. The binding forces involved in TRZ-Tau interaction were also explored using both experimental as well as theoretical docking/molecular dynamics approaches. The results of the current work may open new insights for applying therapeutic potential of TRZ against Alzheimer's disease.

1. Introduction The affected brain in Alzheimer's disease (AD) (which is responsible for 70% of human dementia) displays neuronal loss and nerve cell atrophy along with astrogliosis. In this disease, two types of abnormal structures appear abundantly: neurofibrillary tangles (NFTs) which mostly composed of Tau aggregates and senile plaques (Amyloid beta). However, burden of the disease, the costs of treatments and care is terribly increasing [1,2]. Since the main factor underlying the development and progression of AD is Tau dysfunction/amyloid aggregation. In recent years, several strategies have been proposed to inhibit amyloid aggregation and several structurally unrelated small molecule inhibitors have been shown to prevent and/or reverse Tau fibrillogenesis [3] and to control Alzheimer's disease [4]. The Tau protein as “natively unfolded” or “intrinsically disordered protein, IDP”, with no secondary structure and no stable tertiary structure, stabilizes the assemblies of microtubules [5–7]. This protein is highly soluble and inherently has no propensity to aggregate due to

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presence of many positively charged residues and a relatively small fraction of bulky hydrophobic amino acid side-chains [8–16]. Alternative mRNA splicing was used to generate six Tau isomers in the human brain [17] with different tendencies for amyloid aggregation. In addition to regulating microtubule dynamics, Tau protein also plays vital roles in anchoring intracellular enzymes, and assisting in adjusting vesicular transport [18–20]. Under pathological conditions, Tau hyper-phosphorylation (p-Tau) results in separation of microtubules and accumulation of p-Tau (NFTs formation) leading to neuron apoptosis [21–23], is found in all Tauopathies [24] accompanied with destabilization of axonal MTs [25]. Tau oligomerization triggers neurodegeneration by affecting synaptic operation, as the early hallmark in Alzheimer's Disease (AD) and other Tauopathies [26]. Trazodone (TRZ) is a triazolopyridine derivative [27] acts as the selective serotonin reuptake inhibitor, thereby increasing serotonin levels in the synaptic cleft and potentiating serotonin activity, with antidepressant and sedative properties [28] and also as a serotonin

Corresponding author. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran., Corresponding author., E-mail addresses: [email protected], [email protected] (S. Ghobadi), [email protected], [email protected] (R. Khodarahmi).

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https://doi.org/10.1016/j.abb.2019.108218 Received 20 August 2019; Received in revised form 29 November 2019; Accepted 1 December 2019 Available online 02 December 2019 0003-9861/ © 2019 Elsevier Inc. All rights reserved.

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Fig. 1. (Top, Left). Protein samples were separated by SDS-PAGE (12%) and stained with Coomassie Brilliant Blue R-250. Lane A, molecular weight markers. Lane B and D, SDS-PAGE of eluted pools following purification by Ni2+ Sepharose affinity chromatography. Lane B, the expression was induced with IPTG (1 mM). Lane D, un-induced control. Lane C, SDS-PAGE of crude extract before purification of the protein (Top, Right). Tau fibril suspension absorption spectrum (100 μg/ml) in the presence of CR (A) and CR alone (C). The “corrected Tau + CR” spectrum (D) is obtained by subtracting the spectra of Tau alone (B) from the spectrum of CR in the presence of the protein fibril (A). The difference spectrum (E) obtained from the spectrum of CR in the presence of protein fibril (A) minus the sum of the spectra of Tau alone (B) and CR alone (C). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) (Bottom) Two human Tau isoforms, hT40 (2N4R) and hT34 (1N4R), are represented in the schematic diagram. There are two hexapeptide motifs 275VQIINK280 and 306VQIVYK311 in R2 and R3 shown by black rectangles, in both 1N4R and 2N4R isoforms are necessary for amyloid assembly. P; proline-rich, R; microtubule binding repeat.

2. Materials and methods

antagonist (5-HT2 receptor) and reversible cholinesterase inhibitor [29] used for treatment of major depressive disorders [30]. It has been recently shown that TRZ reduces p-Tau burden [31]. The researchers believe that TRZ in particular as a licensed drug, should now be tested in clinical trials in AD patients [31]. Due to the structural as well as physico-chemical features of TRZ molecule, there is this possibility that this CNS-positive drug (Log P, 2.68), with insignificant anticholinergic effects [32–34], interacts with Tau directly (at PHF6/PHF6* motifs) and reduces its fibrillation extent. The therapeutic dosage of TRZ is 150 mg/day [35,36]. The steady-state plasma concentration of TRZ is 650 ng/ml [37], and the half-life of it is 5–8 h h [38]. Heparin is a typical cofactor that used for in vitro Tau amyloid aggregation [39]. The 4R Tau isoforms are more effective in microtubule binding and aggregate more quickly than the 3R isoforms [40,41], which indicates that isoform expression level is important in Tau aggregation in humans. Furthermore, there is a significantly higher amount of 1 N isoform exists in the brain of adult individuals (54%) in comparison to 0 N and 2 N isoforms (37 and 9%, respectively) [42,43]. Based on the above statements, 1N4R isoform (which employed in the current study, see Fig. 1) has been frequently investigated, in vitro. As stated earlier, the main factor underlying the development and progression of AD is tau fibrillation/oligomerization, so investigating Tau assembly-targeted “small molecule inhibitors” is an emerging and promising approach for AD treatment. In this study, we aimed to obtain some information on the effect of TRZ on Tau fibrillation/oligomerization, inspiring previous studies [44,45]. We investigated Tau amyloidogenesis behavior under laboratory conditions using fluorimetry, circular dichroism (CD), X-ray diffraction (XRD), atomic force microscopy (AFM), and dynamic light scattering (DLS). Intracellular (SHSY5Y cell) Tau oligomerization, under the effect of TRZ was also assessed using flow cytometry technique, and then amyloid cytotoxicity was evaluated using lactate dehydrogenase test and hemolytic assay. The experimental results were also compared with molecular dynamics (MD) outcomes.

2.1. Materials Resin of Ni2+NTA super-flow agarose for efficient purification of the recombinant His-tagged proteins was purchased from QIAGEN. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Sigma-Aldrich (Munich, Germany). Luria Bertani Broth was purchased from HiMedia (Einhausen, Germany). Heparin (average molecular mass of 15000 Da) was obtained from Celsus Laboratories (Cincinnati, OH). Thioflavin T (ThT) was purchased from Sigma-Aldrich (Munich, Germany). ABN454, Tau oligomer-specific antibody, T22 was obtained from EMD Millipore, (USA). All other chemicals used in this experiment were analytical grade and were used as received without any further purification. 2.2. Protein expression and purification Tau protein was expressed and purified as described previously by Rankin et al. [46]. The pET21-Tau plasmids contained 1N4R Tau isoform were transformed into BL21 E. coli and grown in Luria Broth + ampicillin until an OD600 of 0.6–0.7 was reached. Then, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added (1 mM) and cultures were incubated for 2 h. The cells were centrifuged (at 12,000 g) and re-suspended in the lysis buffer (50 mM Tris-HCl, 30 mM Imidazole, 100 mM NaCl, 2 mM PMSF, 5 mM DTT, pH 8). Purification was done using affinity chromatography through a Ni2+NTA Sepharose (Qiagen, Valencia, CA, USA) in which protein binds to Ni2+ when passed through the affinity column before being released in the elution buffer (50 mM Tris-HCl, 250 mM Imidazole, 100 mM NaCl, pH 8). The purity of Tau was confirmed by SDS-PAGE gel electrophoresis, and then protein was aliquoted and stored frozen at −20 °C, until use. 2.3. Congo red binding assay Congo Red (CR) binding assay was performed according to previously described method by Nilsson et al. [47] with minor 2

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modifications. The CR stock solution (100 μM) was prepared in potassium phosphate buffer (20 mM, pH 7.4), then filtered. Tau test sample was mixed with CR solution to yield the final concentration of 10 μM CR and 100 μg/ml of Tau fibrils. The CR-Tau fibril mixture was incubated at room temperature for 15 min prior to spectral analysis over a range of 400–700 nm.

log (F0 − F )/F = log K + nlog [Q]

where K is the binding constant and n is the number of binding sites. The standard enthalpy and entropy change of the interaction were obtained using the van't Hoff equation:

LnKb = −ΔH 0/RT + ΔS 0/R

The Thioflavin-T (ThT)-based fluorimetric method was used to evaluate the TRZ inhibitory effect on Tau aggregation [48], with minor modification. Since Xue et al. [49] reported that ThT fluorescence intensity correlates linearly with amyloid concentration, thus ThT can be reliably used as a quantitative probe to measure amyloid concentration. Purified Tau protein was dialyzed against dialysis buffer (50 mM TrisHCl, pH 7.5) and incubated at final concentration of 50 μM with 5 μM heparin, either alone or with various concentrations of TRZ (0, 10, 30 and 50 μM) at 37 °C. PHF6 (305SVQIVYK311) is one of the hot-peptides in Tau protein aggregation. FVQIVYH peptide is analogue of PHF6 with higher hydrophobicity that is able to accumulate in the absence of any additional inducer [50]. This peptide is an important segment of Tau protein for fibril formation [51]. Therefore, we evaluated the inhibition effect of different TRZ concentrations (10, 30 and 50 μM) on FVQIVYH peptide (50 μM) aggregation, in the absence of any inducer. Stock solution (1 mM) of ThT was prepared in 50 mM Tris-HCl buffer, pH 7.5. The assay solutions (final ThT concentration; 20 μM) were excited at 440 nm and emission was recorded at 485 nm.

ΔG 0 = ΔH 0 − TΔS 0

(4)

(5)

2.6. CD measurements Quantitative nature of CD spectroscopy makes it as a very useful tool for studying the protein-ligand interactions since the amount of change in the CD spectrum of a protein is directly proportional to the extent of changed protein due to ligand binding [56]. Protein solution was incubated at 37 °C for 120 h to aggregate. The changes in the CD spectrum of Tau was monitored by a J-810 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a peltier system for controlling the temperature (at 25 °C) using a quartz cell with 1 mm path length in 50 mM Tris–HCl, pH 7.5. Far UV-CD spectra of Tau protein with and without TRZ were recorded over a wavelength range of 190–260 nm. The results were presented as mean residue ellipticity, MRE, (deg cm2 dmol−1) according to the following equation: MRE = ϴobs/ (Cpnl × 10)

2.5. Fluorescence quenching and Kb calculation

(6)

where ϴobs is the measured ellipticity (mdeg) at specified wavelengths, Cp is the Tau molar concentration, n is the number of Tau amino acid residues and l is the length of the CD cuvette in cm [57].

Steady-state fluorescence emission spectra of samples were recorded by a fluorescence spectrophotometer (Cary Eclipse, Varian) in a 10 mm quartz cuvette with a jacketed cell holder in which temperature was controlled by an external thermostated water circulation system. For determination of fluorescence quenching mechanism, the emission spectra was scanned from 290 to 350 nm at 1 nm intervals, while excitation wavelength was 275 nm, using 5 and 10 nm for excitation and emission slit widths, respectively. To quantify the binding constants of TRZ to Tau, a 2.0 ml solution of 30 μM Tau was monitored in the presence of different concentrations of TRZ with trace syringes (to give a concentration from 20 to 50 μM). Fluorescence quenching experiments were studied at four different temperatures (280, 290, 300 and 310 K) with recycled water keeping the temperature constant. The appropriate blanks corresponding to the Tris–HCl buffer solution were subtracted to correct the background of the fluorescence spectra. To reduce the inner filter effect, both intensities of the fluorescence arising from excitation light absorption and emission light re-absorption were corrected using the following equation [52,53].

2.7. Dynamic light scattering (DLS) analysis After incubation of the purified Tau (30 μM) either alone or in the presence of 50 μM TRZ (at 37 ∘C for 48 and 96 h, the needed time for oligomerization, and plateau reaching steps, respectively), their ThT fluorescence was measured. After reaching the ThT fluorescence to the plateau region, the samples were filtered with a 20 nm syringe filter (Whatman, Maidstone, UK). Afterwards, the size and the size distribution of the oligomers were investigated using a Malvern Nano ZS instrument equipped with a peltier temperature controller and DTS software (Malvern Instruments, UK). Polystyrene cuvettes with 1 cm path length were used for DLS experiments. The results were analyzed by Zetasizer software (V6. 12) and presented as a mean of three independent batches for each sample. 2.8. X-ray diffraction analysis

(1)

here, the corrected and observed fluorescence intensities are represented as Fcor and Fobs, respectively. While, the values of TRZ absorbance at excitation and emission wavelengths are represented as Aex and Aem, respectively. The quenching rate constant values were obtained using the SternVolmer equation:

F0/ F = 1 + K SV [Q] = 1 + kq τ0 [Q]

R = 8.31 mol−1K−1

where Kb is the binding constant at various temperatures (T = 280, 290, 300, and 310 K) and R is the universal gas constant. The standard free energy change of the binding process was estimated using the well-known Gibss equation:

2.4. Fibril formation by Tau and the modified PHF6: The effect of TRZ

Fcor = Fobs [antilog (Aex + Aem )/2]

(3)

The signal that originate from a single molecule is too weak to measure, but the scattering signal arising from crystals (that contains ~1015 identical macromolecules periodically ordered in three dimensions) is easy to detect [58]. So, for X-ray diffraction analysis, the crystals of 20 μl samples (protein solutions with and without the drug) were prepared by drying on the glass, The prepared crystals were studied by scanning rate of 40/min, using a Rigaku X-ray Powder diffractometer with Cu anode (Cu-Kα radiation, λ = 1.54 Å) in the range of 50-600 and at 40 keV.

(2)

In the above equation, F0 and F are the fluorescence intensities in the absence and in the presence of the drug. kq is the quenching rate constant. τ0 is equal to 1 × 10−8, which indicates the half-life of the fluorophore in the absence of the drug [54], KSV is the Stern-Volmer constant and [Q] is concentration of the quencher. The modified Stern-Volmer equation (Eq. (4)) was used for evaluation of the binding constant [55]:

2.9. Atomic force microscopy of samples Each sample was prepared by incubating 200 μl (20 μM of Tau plus 5 μM of heparin) with and without TRZ (50 μM) for 144 h. Then, 10 μL of the sample poured on freshly cleaved mica. After 5 min, the surface 3

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centrifugation at 1000g for 10 min and washing three times with PBS solution. Then, 0.5 mL of the washed blood cell suspension was added to 9.5 ml saline solution (0.9%) as a stock solution. After the fibril formation was confirmed by ThT assay, the different samples [Tau samples (30 μM) were incubated at 37 °C with and without TRZ (50 μM) in two different times (48 and 96 h)], TRZ alone (50 μM) and buffer, as a negative control were mixed with cell suspensions separately. The mixture was incubated for 1 h at 37 °C and then centrifuged at 3000 g for 10 min. The supernatant was removed by suction and the absorbance of the samples was measured on the standard micro titer plate by ELISA reader at 540 nm.

of mica washed gently twice with deionized water, allowed to dry for 10 min. Mica exposes a negatively charged surface that protein containing positive charges can be mounted on it. Imaging was attained at room temperature using the non-contact mode, dual scope/raster scope C-26 atomic force microscope with tip radius ˂ 10 nm and resonance frequency of 258 kHz. 2.10. Induction of Tau aggregates in SH-SY5Y cells 2.10.1. Determination of intracellular Tau oligomers by flow cytometry Flow cytometry was applied for detection of induced Tau oligomers using the Tau oligomer-specific antibody, T22 (EMD Millipore, USA) [59]. The human neuroblastoma cell line SH-SY5Y was cultured in DMEM/F12 medium containing 10% FBS and 1% Pen Strep, (all from Gibco, Canada) at 37 °C with 5% CO2 in a humid atmosphere. SH-SY5Y cells were plated in culture flask. For detaching, trypsin-EDTA added to confluent SH-SY5Y cells and the flask was left for 2–3 min at 37 °C, then re-suspended in full medium, counted, and seeded into 6-well plates at a density of 30 × 104 cells/well. The day after, cells were exposed to 100 μM CR for five days for induction of Tau aggregation according to Lira-De Leon et al. method by minor modifications [60]. At the third day, half of the total media volume was replenished with the same concentration of CR solution. Afterwards, the cells were exposed to TRZ (50 μM) and incubated for 48 h. After removing the peripheral environment, the cells were washed by PBS, followed by detachment with 0.25% trypsin-EDTA and collected. Then, permeability of cells were done by 0.2% Tween-20, resuspension in PBA [phosphate-buffered saline (PBS) 1X, BSA 0.1%] and fixed with 4% paraformaldehyde for 10 min at room temperature. To prevent nonspecific interactions, the cells were incubated in 1X PBS, 10% normal goat serum and 0.3 M glycine for 30 min. Afterwards, prepared T22 antibody in PBS including 0.2% Tween-20 and 1% BSA (1:500) was added to the cells and incubated for overnight at 4 °C. In the next step, to remove the first antibodies that were not bonded, the rinsing was done again and the cells were collected. In the following FITC-sheep, anti-rabbit secondary antibody (1:500) was added on the cells and incubated in the dark room for 1 h. After washing with PBS and slowly vortexing, the cells were monitored by flow cytometer blue beam and fluorescence microscopic imaging (Section 2. 9. 2). The obtained data were analyzed with FlowJo version 7.6.1 software (Tree Star, Ashland, OR, USA).

2.12. Colorimetric LDH assay The cytotoxicity was monitored using LDH assay as described by Kaja et al. [64] with minor modifications. The SH-SY5Y cells (cultured according to Section 2.9.1) were transferred to sterile 96 well plates at a density of 10,000 cells/well and 3 wells per treatment. The day after, the medium was replaced with serum free media and the cells were treated by the aliquots of the aggregation products of Tau protein either alone or with 50 μM TRZ taken at three different times of incubation (t = 0 of incubation when the majority of the proteins are monomers, the end of the lag phase which is supposedly rich in oligomers (48 h) [65,66] and 96 h of incubation of Tau for mature fibrils) under fibrillation condition that mentioned in Section 2.3. The toxicity was assessed by adding samples to the cells at a final concentration of 5 μM and incubation for 48 h. After 48 h incubation, the effect of samples on cell viability was evaluated using LDH assay kit protocol (LDH assay kit, Roche, Germany). The absorbance of converted dye in LDH assay was measured at 495 nm with background subtraction at 630 nm. The percentage of viable cultured cells exposed to differing samples was reported. Each concentration was tested in three independent experiments. 2.13. Molecular dynamics (MD) simulation Herein, the molecular dynamics simulation was carried out to study the molecular behavior of Tau protein in the presence of the ligand (TRZ), and especially for providing of the coordinates of Tau protein required in docking and the binding pocket finding analysis. To save structures of Tau protein, MD simulation was applied utilizing GROMACS simulation package version 5.1.4 [67] with Gromos 54a7 force field under an Intel Core i7 Extreme Edition under Centos Linux 6.8. The protein was first located in the center of the cubic system following by the randomly adding of solvent water molecules in the SPC/E model and 50,000 steps of steepest descent energy minimization while a time step of the 2 fs applied for integration of the equation of motion. Afterwards, the system was equilibrated under NVT and NpT ensembles for 500 ps in each one. The temperature was controlled near 300 K by Berendsen algorithm [68] with a time constant of 0.1 ps, besides the pressure was controlled at 1 bar by Parrinello-Rahman algorithm and the temperature coupling time constant of 0.1 ps and 0.2 ps of the pressure coupling time constant [69]. A periodic boundary was used in the x, y and z directions while LINear Constraint Solver algorithm [70] was performed to constrain all bonds and short-range van der Waals interactions in Lennard-Jones potential was set at 1.4 nm. A Particle-Mesh Ewald algorithm [71] was assigned to deal with longrange electrostatic interactions in Coulomb potential energies with the real space contribution to the Columbic interactions truncated at 0.9 nm. The initial velocity of particles was generated according to Maxwell distributions. Finally, 100 ns of the MD simulation was produced while extracting coordinates every 10 ps of time intervals. The structural indicators of the system Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of gyration (Rg) and Solvent Accessible Surface Area (SASA) were calculated using modules of the gmxrms, gmxrmsf, gmx gyrate and gmxsasa,

2.10.2. Fluorescence microscopic imaging The intensity of DNA fluorescence at 525 nm, excited at 495 nm, was aggravated by the addition of acridine orange (AO) to intercalate into the DNA base pairs [61]. In this work, AO was used as a sensitive probe for DNA microscopic study, for measurement of the amount of survived cells. At first, a stock AO solution (1 mM) was prepared (100X, dissolved in 1 mL of 95% ethanol and 49 mL distilled water). Working solution 1X was used by diluting in PBS. All of the solutions were prepared freshly. 25 μl cell suspension of SH-SY5Y with 5 × 104 cells/ mL was added to 25 μl of AO solution and gently mixed. Afterwards, 20 μl of the final solution was taken and placed underneath the coverslip over a hemocytometer slide. The viability of the cells was investigated by switching mode of excited to fluorescence mode (using 495 and 525 nm as primary filter secondary filters, respectively). Live cells were green in the presence of AO [62]. 2.11. Hemolytic effects of Tau fibrils is attenuated in the presence of the drug Erythrocyte hemolysis was performed, according to the previous reported method [63] with minor modifications. Briefly, whole human blood was collected in EDTA-containing Vacutainer blood collection tube, followed by separation of erythrocytes (RBC) from fresh blood by 4

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3. Results and discussion

respectively, from GROMACS 5.1.4 simulation package. All of the computations were done at last 80 ns of the simulation time where the system RMSD calculations deal with the plateau. For validation of the drug effect on the Tau protein structure and the study of the protein molecular dynamic behavior with the drug, the next simulation system was organized using randomly adding of the 10 drug molecules in the presence of an individual Tau protein. This computation was planned using the same condition of the first part.

3.1. Protein purification The purity of expressed Tau (in E. coli, BL21) was checked during the purification steps and finally confirmed using SDS-PAGE and western blotting methods. The crude extract from BL21 contained several bands (analyzed in a CBB-R250 stained SDS-PAGE gel) is shown in Fig. 1, lane C. After purification on Ni2+-affinity chromatography, the eluted fraction migrates as a single band at ~53 kD, correlated to 1N4R Tau, confirming its purity (Fig. 1, lanes B and D). Immuno-detection of 6 × His tagged end by HRP conjugated rabbit polyclonal antibody, showed the existence of monomeric protein (data not shown).

2.13.1. Binding pocket analysis Protein binding site analysis was done along the trajectory where the system RMSD calculations reach to plateau at the time period of 20–100 ns using MDpocket software [72]. 8000 conformations were extracted from the simulation and the structures were subjected to MDpocket package binding site searching on the structural ensembles to visualize pocket frequently maps.

3.2. CR binding studies Tau amyloid formation was verified using Congo red (CR) binding assay. The individual and combined spectral properties of fibrillar Tau and CR are displayed in Fig. 1, right. CR spectral shift is dependent on the protein aggregation state [74]. Since amyloid fibrils possess substantial light scattering (registered as pseudo-absorption or turbidity in absorption spectroscopy) in addition to strong absorption in UV region, (absorbance) spectra of a suspension of Tau fibrils alone (or scattering contribution of Tau fibrils alone), suspension of Tau fibrils in the presence of CR, and of CR alone were independently recorded at 400–700 nm wavelength range (Fig. 1, right). To eliminate the contribution from scattering and absorption of the Tau fibrils, the difference spectrum (as pure “CR” spectrum, in the presence of amyloid fibrils; curve E in Fig. 1, right) is obtained by subtracting the spectra of Tau fibrils alone and CR alone from the spectrum of CR in the presence of the protein. As indicated in this figure, the absorption spectra of CR on binding to the Tau amyloid aggregates showed a red shift of the maximum absorbance of CR from ~490 nm to ~520 nm (compare curves D/E with curve C), which is a characteristic of amyloid aggregation [75,76].

2.13.2. Molecular docking analysis To overcome the structural flexibility of Tau protein, docking analysis was planned along the trajectory of the simulation in the last 80 ns of the equilibrated area. The receptor coordinates were extracted from the trajectory. In the receptor, water molecules were removed from the structure, polar hydrogen atoms were added to the protein and Gasteiger partial charges were computed as well followed by saving the structure of the ligand and receptors in PDBQT format. Then, the affinity energy values (kcal/mol) for the ligand in each conformation of Tau was calculated using AutoDock Vina software [73]. Two simulation systems were applied in the current research. In the first one, through 100 ns MD simulations for the Tau protein, an ensemble of the molecular conformation of the Tau was extracted to neglect the structural fluctuations of the protein. The Tau protein has absolutely high flexibility during the simulation. Therefore, the simulation continued enough until reaching statistically adequate structures for reliable computation and 8000 conformations were saved during the MD simulation. However, the RMSD is a one-dimensional property in a multi-dimensional simulation system and may not be a characteristic of a converged system, but the differences between the extracted conformations were apparently suitable to convince us for the next calculations.

3.3. TRZ attenuates Tau Fibril formation: Tight binding to the protein as prerequisite of inhibitory activity of the drug As shown in Fig. 2 A, in the absence of the drug and in the presence of different concentrations of TRZ, successive incorporation is going-on so that finally amyloidogenesis stops at the plateau phase in which maximal amount of protein monomers was assembled into β-sheet amyloid [77]. Measurements to probe the in vitro amyloid aggregation kinetics of Tau protein with and without TRZ show that the drug affects Tau amyloidogenesis, monitored by ThT fluorescence assay. As indicated in Fig. 2 A, at 50 μM TRZ, less than 50% of the Tau fibrillation (based on ThT fluoresence at plateaus or aggregation extents) was inhibited by

2.14. Statistical analyses The SPSS software version 16.0, SPSS, Inc., Chicago, IL, (and Graph Pad 6) was applied to perform statistical analysis for the obtained data. Statistical comparisons between different groups were made using oneway analysis of variance (ANOVA). Otherwise stated, results were expressed as the means and standard error of the mean (mean ± SEM), and P values less than 0.05 were considered statistically significant.

Fig. 2. (A). Effect of different concentrations (10, 30 and 50 μM) of TRZ on the kinetics of Tau (30 μM) fibril formation determined by ThT fluorescence monitoring at 37 °C in 50 mM TrisHCl buffer, pH 7.6 and 7.5 μM heparin. (B) Analysis of the Peptide (50 μM) fibril formation, at various time intervals (0, 4, 24 h), in the absence or presence of different concentrations of TRZ (10, 30 and 50 μM) [44]. Three independent experiments were considered to calculate the spread in the data shown by the error bars. Some error bars (SD) were marginal and thus are masked by the symbols/columns.

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the slope of the straight line is equal to n and the Y-intercept equals to log K. The obtained values of n in the interaction of TRZ with Tau is approximately equal to one, indicating that there is only an effective binding site on Tau for TRZ binding. The binding constant of TRZ to Tau protein is similar to that obtained previously for curcumin, with a high tendency to binding [44]. Binding constants at various temperatures are in the range of ( × 105 M−1). The results of the van't Hoff plot (data not shown) indicated that hydrogen bonding and van der Waals interactions play an important role in Tau interaction with TRZ since both ΔS0 and ΔH0 are smaller than zero [79]. The estimated ΔG0 and ΔH0 of the interaction between TRZ and Tau are both negative, indicating that the interaction is spontaneous, exothermic, and enthalpy driven. These results are consistent with observed slight decrease in Kb values with temperature increment and guarantee relatively tight binding of TRZ to the native/monomeric form of protein, as a prerequisite for interfering with Tau fibrillation.

the drug. As indicated in Fig. 2A, TRZ has affected both the elongation phase (note and compare the slopes of the curves; as amyloid aggregation rates) and, to a lesser extent, nucleation step of Tau amyloidogenesis. It seems that elongation beginning occurs after ~30–40 h of incubation, so, it appears that nucleation step (lag phase) has been slightly affected (and or elongation has been postponed) in the presence of 50 μM TRZ. So, the drug appears to interfere with the production of mature fibrils and affect formation of amyloid precursors/oligomers as well. In this way, the overall aggregation extent (ThT signal at t = 96 h/110 h) will be affected by TRZ. In other word, less fibril or less β-structure is formed in the presence of the drug. A key role in Tau amyloid assembly is the formation of β-sheet structure driven by two hydrophobic short hexapeptide motifs in the second and third repeat of Tau, PHF6* 275VQIINK280 (in R2) and PHF6 306 VQIVYK311 (in R3, see Fig. 1), and it has been speculated that the interaction between these two regions gives rise to the Tau fibrillation and eventually unique PHF morphology. The PHF6 segment in Tau protein has been shown to be important for fibrillation of the full-length protein and it alone forms fibers with biophysical properties similar to full-length Tau fibrils. The modified heptapeptide, FVQIVYH, as more hydrophobic analogue of PHF6 (the wild peptide ‘SVQIVYK’ changed to ‘FVQIVYH’), contains the “VQIVY” amyloidogenic segment but with much higher amyloid aggregation propensity [44]. As described above and in our earlier work [44], SVQIVYK (PHF6) peptide segment of Tau is able to fibrillize in the absence of any added inducer compound. In this assay we evaluated amyloid aggregation of its analogue (FVQIVYH), in the absence and presence of TRZ. It was found that TRZ is effective in inhibiting peptide fibril formation (Fig. 2 B). Therefore, TRZ probably hinders the formation of Tau aggregates by binding to these amyloidogenic segments, and controlling the collision of these peptide fragments. Effective TRZ-Tau interaction, especially at two hydrophobic/amyloidogenic segments of the protein, (or tight binding of TRZ to the protein) is an important prerequisite of successive inhibition of Tau amyloid assembly by the drug. As indicated in Table 1 and Fig. 3, Ksv and the quenching ability (kq) of the drug decreases with increasing temperature. Fluorescence quenching is affected by various factors such as energy transfer, ground-state complex formation and collisional quenching. The two kinds of quenching mechanisms (dynamic and static) differ from each other as the consequence of their different dependency on temperature [78]. Due to increasing diffusion coefficient for dynamic quenching, quenching constant increases with increasing temperature. While in static quenching, rising the temperature results in the complex instability and thus reduces the quenching constant. As shown in Table 1, along with the increase in temperature, the observed quenching is reduced (Ksv and kq), which indicates that the mechanism is static type. It is indicated that quenching accomplished by the formation of a ground-state complex, which becomes unstable as the temperature rises and so the amount of quenching is reduced. The therapeutic value of each drug depends on its binding strength, which affects its stability and toxicity. Using the fluorescence quenching data, the number of binding sites and the values of binding constant were obtained from equation [4]. In the plot of log (F0–F)/F versus log [Q],

3.4. CD analyses The systematic changes to both the magnitude (i.e. chirality of the signal, either positive or negative) and the characteristic shape of the detected far UV CD signals give insight to what occurs to the proteins' secondary structure. We used Far-UV CD spectroscopy to gain insight into secondary structural characteristics of Tau amyloid aggregates. The far UV CD spectrum of fresh Tau sample in the absence of additives is characterized by a large negative minimum band near 200 nm, as characteristic of all intrinsically disordered proteins. For the Tau sample incubated in the presence of the inducer (heparin), appearance of a negative band at ~218 nm indicates a β-type conformation of the produced fibrils (Fig. 4). Also, as indicated in this figure, adding of the inhibitor (TRZ) to the Tau sample decreased the intensity of the band at ~218 nm, clearly indicating the reduction in the β-sheet content of the treated samples. The change of the protein CD signal was small confirming a moderate ability (inhibitory effect) of TRZ to alter secondary structure contents of the β-rich Tau fibrillar aggregates. The possibility of the drug-induced disintegration of the fibrils to the oligomeric populations will be also discussed. 3.5. DLS measurements To gain insight into the size of the amyloid aggregates present in solution, samples incubated in the absence and presence of TRZ were further analyzed using DLS. Based on the CD data, Tau samples incubated with the drug, contained a greater amount of β-sheet content per unit volume than freshly dissolved protein (or Tau incubated in the absence of heparin), but a lower β-content than fibrils. TRZ, as a fibrillation-modifying compound, may mainly affect/impede late stages of Tau fibrillogenesis and alter typical fibrillar assemblies to the other types of aggregates (including amorphous aggregates) with lower βcontent; on the other hand, the drug, as an inhibitor of early stages of amyloidogenesis pathway, prevents the formation of oligomeric intermediates and smaller sized intermediates are populated. A mixed mechanism of action is either likely. The size distribution of Tau species with the progress of heparin-

Table 1 Stern–Volmer quenching constants, thermodynamic parameters, binding constants (Kb) and number of binding sites (n) for binding of TRZ to Tau at different temperatures. TRZ

T (K)

K SV (M−1)

R2

× 10−4 280 290 300 310

3.82 2.76 1.57 0.66

0.99 0.99 0.94 0.97

kq (M−1S−1)

Kb (M−1)

× 10−12

× 10−5

3.82 2.76 1.57 0.66

5.19 4.77 1.63 0.80

n

1.3 1.3 1.2 1.2

6

R2

0.99 0.99 0.99 0.99

ΔH 0

ΔS 0

ΔG 0

(kJ mol−1)

(J mol−1 k−1)

(kJmol−1)

−45.29

−0.05

−30.85 −30.33 −29.82 −29.30

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Fig. 3. (A) Stern-Volmer and (B) the plots of log (F0–F)/F versus log [Q] at different temperatures (280, 290, 300 and 310 K) in the presence of TRZ. Stern-Volmer quenching constant (Ksv) was obtained from the slope of Stern-Volmer plots. Data shown are representative examples of three similar independent experiments, and standard deviations were almost within 5% of the experimental values.

3.6. XRD analysis XRD is the grand technique for deciphering the atomic structure of proteins. This technique supplies a good way to directly examine crossβ structures because it provides direct information on the molecular structure. There are two typical types of diffraction patterns in the structure of β aggregation [81,82]. A diffraction pattern is the distance between hydrogen bonds between β-strands (4.7–8 Å, denoted by peak of 2θ around 20°). The other pattern of diffraction is related to the distance between the β-sheets, which is dependent on the size of amino acid side chains between the β-sheets (10–12 Å, denoted by peak of 2θ around 10°) [83]. According to the Brag's law: nλ = 2dsinθ, a constant wavelength with n is also a constant number, so if d decreases, 2θ increases. As shown in Fig. 5D, as plots of scattering intensities versus the scattering angle 2θ, XRD pattern of (TRZ) untreated Tau peptide sample exhibited two peaks, corresponding to d-spacings of ~4.7 Å (denoted by peak of 2θ around ~20°) and ~10.4 Å (denoted by peak of 2θ around ~10°). Since formation of Tau fibrils is driven by two hydrophobic short hexapeptide motifs, PHF6/PHF6*, with no doubt the above mentioned peaks are attributed to the crystal-like structures formed by these amyloidogenic segments. The sample, incubated in the presence of TRZ (Fig. 5C) displayed clearly discernible pattern of scattering at the same peaks (2θ around ~20° and ~10°), characteristics of cross-β sheet structure, but with much lower intensities, confirming (Tau) fibrillation inhibitory potency of TRZ. A ~50% decrease in the peak (2θ around ~20°) intensity may confirm: 1) TRZ tight binding and masking the available amyloidogenic segments of (amyloid) aggregation-prone Tau intermediates, mainly at early stages of Tau fibrillation 2) effective intercalation of the drug between stacked hydrophobic side chains of “hydrogen-bonded βstrands of amyloidogenic segments” within intermediate-sized oligomers/protofibrils in a kind of “butter-knife” mechanism, allowing a zipper-like rupture of extended beta-sheet along the fibril axis. This would be a likely mechanism of fibrillation attenuation at late stages of Tau fibrillization (see Scheme 1). Additionally, the peaks were not completely disappeared in the presence of TRZ which possibly reminiscent of incomplete dismantling of Tau fibrils and formation of βrich (ThT positive) smaller (proto-) fibrils as well as some (non-toxic) oligomers, an observation that is in agreement with ThT, CD data as well as AFM images.

Fig. 4. Far UV-CD spectra of Tau with and without the drug; TRZ. Far-UV CD spectra, recorded for (A) fresh Tau (B) Heparin-Tau and (C) TRZ-Heparin-Tau at 25 °C. The Tau concentration was 20 μM in Tris-HCl buffer (50 mM, pH 7.6). TRZ and Heparin concentrations were 50 and 5 μM, respectively.

induced fibrillogenesis in the absence/presence of TRZ was monitored. Coinciding with the period of fast ThT fluorescence increase (Fig. 2A, t = 48 h), Tau molecules almost contribute to elongation phase and aggregate size temporally increased showing a mean diameter around 800–900 nm as the likely result of the formation of the first mature amyloid fibrils. The size distributions obtained in the presence of the TRZ confirmed the inhibitory potential of the drug. Even though DLS yields qualitative, not quantitative results [80], the mean diameter/ particle size of the aggregates formed in the presence of TRZ slightly decreased, compared to those of the un-treated samples (Data not shown). Additionally, the DLS interpretation for untreated fibrils can be tricky (only the hydrodynamic size and not a direct fiber length can be observed) and since the scattering intensity of a particle is proportional to the 6th power of its diameter, the DLS results of TRZ-treated samples may suggest the existence of a population of amorphous aggregates with the same mean diameter. Thus, given the limitations of DLS to morphologically describe less prevalent, non-spherical particles, the amyloidogenic samples were also characterized using AFM. Therefore, although TRZ reduced β-content of Tau assemblies (see CD data), the large aggregates are still formed in the presence of the drug similar to the case of size distribution obtained when the protein is allowed to fibrillate in the absence of TRZ. This observation, with partial accordance with ThT data, may suggest the partial inefficiency of TRZ in interfering with the early stages of Tau fibrillation process, but as indicated by CD, ThT and XRD data of this communication, TRZ has an acceptable potential to impede fibrillogenesis.

3.7. Characterization of fibril morphology Atomic force microscopy (AFM), as an excellent tool for investigating amyloid aggregates, has been extensively utilized to characterize aggregate morphology, and mechanical properties. Here, the fibril formation of Tau in the absence of TRZ and morphological changes of the amyloid assemblies in the presence of the drug were verified using AFM. Incubation of Tau with heparin, in the absence of 7

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Fig. 5. (Top). AFM images of Tau (20 μM) in the presence of heparin (5 μM) (A), and in the presence of heparin (5 μM) + TRZ (50 μM) (B). Reduced fibril formation in the presence of TRZ is quite obvious. (Bottom). XRD patterns of Tau + heparin with (C) and without (D) TRZ were recorded after 108 h incubation at 37 °C in TrisHCl buffer (50 mM, pH 7.6) using a Rigaku Miniflex X-ray diffractometer in 2θ ranging from 5∘ to 60∘.

immunospecific detection of fluorescein isothiocyanate (FITC) fluorescence signal. During immunospecific detection of oligomers in SH-SY5Y cell line, a significant increase in FITC signal was observed in CR-induced cells, in the absence of TRZ (Fig. 6B, E), compared to that of control sample with no CR induction (Fig. 6A, E), confirming Tau-oligomer production under the effect of CR treatment. Upon treatment of CR-induced cells with TRZ, FITC signal decreased almost like the “uninduced control” signal (Fig. 6C, E), reminiscent of significant reduction of intracellular oligomers. It is noteworthy that T22 (Tau oligomerspecific antibody) does not show any significant reactivity for monomeric Tau, but some signal is observed in Tau fibrils, possibly due to the cross-reactivity of the antibody or the presence of oligomers in the preparations. Previous studies have confirmed that inhibiting intracellular amyloid aggregation/oligomerization of α-synuclein [86] and dipeptide repeat (DPR) [87] reduces cytotoxicity [88]. Given the reduced FITC fluorescence in TRZ-treated samples, it can be concluded that TRZ reduces intracellular Tau fibrillation through reducing the population of the resulting intermediate-sized toxic oligomers and thus decreases cell death. Based on the obtained data, it seems that penetrating (intracellular) TRZ molecules (interact with Tau monomers and) interfere with nucleation stage, and with the formation of such early nuclei, prevent the onset of oligomerization and formation of subsequent assemblies, thus causes diminished cytotoxicity. As can be deduced from Fig. 6 (A-C), the number of normal cells in the un-induced control and TRZ-treated samples is approximately equal

TRZ, led to formation of Tau fibrils, corroborating the ThT fluorescence data (see Figs. 2 A and Fig. 5). While, incubation of Tau with TRZ gave ThT-positive (based on Fig. 2) small/short oligomeric assemblies with an appearance similar to amorphous oligomeric aggregates [84]. Taking AFM, ThT, CD and DLS data into account, the protein sample incubated with the drug is still capable of light scattering confirming the presence of (smaller/amorphous) protein aggregates, but with lower content of β-structures. So, it is logical to consider TRZ as a moderate fibrillation inhibitor rather than an aggregation suppressor. The possible cytotoxicity of these species will be assessed. Recall that oligomeric species may even cause toxicity and disease, while mature fibrils do not have this ability [85]. 3.8. Effect of TRZ on the intracellular Tau amyloid aggregation/ oligomerization in SH-SY5Y cells Increasing evidence indicates that oligomeric species of Tau formed as intermediates during the fibrillation process are substantially more toxic to neuronal cells than are the mature fibrils. There is possibility that TRZ reduces population of oligomers at early stages of Tau amyloid aggregation, and mitigates the resulting toxicity to neuroblastoma cells. At the same time as induction of Tau amyloid aggregation in the SHSY5Y cells via exposure to Congo Red, and to determine the amount of induced intracellular Tau oligomers under the effect of TRZ, the fluorescence signals of the treated sample as well as respective controls were investigated using flow cytometry method through 8

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Scheme 1. An initiating event in Tau aggregation may be inducer-mediated conformational change and/or covalent modification (phosphorylation, truncation), facilitating conversion of the native Tau protein to an amyloid aggregation-prone conformation. Globular intermediates (oligomers) are formed from these competent monomers and then protofibrilar structures are assembled. Amyloid fibrils can then form through association of protofibrillar intermediates. Through partial masking of the amyloidogenic segments of the aggregation-prone Tau monomers, early inhibition of Tau fibrillation occurs and TRZ attenuates the formation of potentially toxic globular intermediates (oligomers). Moderate inhibition of “oligomer conversion to protofibril” and “protofibril conversion to fibril” steps by the drug is also likely. If TRZ affect later stages of Tau fibrillation, it may result in accumulation of toxic oligomers, off-pathway globular intermediates and even large amorphous aggregates. All of these species have β-structures but with a lesser content, compared to mature fibrils.

nevertheless much lower than the hemolysis seen with Cell Lysis (Triton X-100) Solution. Consistently with the ThT assay and SH-SY5Y cellbased (flow cytometry) studies, and as shown in Fig. 8, TRZ attenuates Tau amyloid-induced membrane disruption of the withdrawn samples at the mentioned time intervals. After 48 h incubation, Tau amyloid aggregates induced a hemolysis rate of 60 ± 1.5% which was reduced by 40 ± 2% under the influence of the drug. Also, TRZ slightly reduced hemolysis rate of mature Tau amyloids (t = 96 h), from 30% to 25%. However, no significant hemolysis of RBCs was detected in the presence of buffer alone or TRZ alone, as negative controls. Moreover, the interaction of preformed β-rich assembled fibrils (48 h incubation under the amyloidogenic conditions, then treated with TRZ) with RBC membrane was investigated. The results showed that TRZ was unable to mitigate hemolysis of preformed Tau fibrils that confirms that the drug may not be a β-sheet breaking compound and, to clarify it, further evaluations should be made using different techniques. Membrane disruption by intermediate-sized toxic amyloid particles (oligomers) is a universally accepted fact. Only the toxic aggregates have sufficiently high structural plasticity and hydrophobic surface to penetrate the cell membrane. Hemolysis data may partially confirm the reduced population of oligomers in the presence of the drug. Furthermore, regarding these data and the earlier results of the current study as well, it appears that TRZ reduces amyloid-induced hemolysis by reducing intermediate-sized amyloid particles (Scheme 1) rather than by interrupting amyloid aggregate/intermediate interactions with the cell membrane.

(98.7% and 98%, respectively), while in untreated (CR-induced, 6B) samples, the number of normal cells decreased (93.9%). That is, about 6% of the cells have changed in terms of cell shape and dimensions (side scatter channel (SSC)) as well as in terms of density (forward scatter channel (FSC)) compared to that of control. However, TRZ treatment (via reducing toxic oligomers) prevents changes in density and subsequently cellular deformation, which may prevent cell death in this way. Taking these results and earlier (ThT, XRD and AFM) data into account, TRZ may be recognized as a moderate rather than potent fibrillation inhibitor that either is capable of interfering with Tau oligomerization. 3.9. Fluorescence microscopy investigations As can be seen in Fig. 7C, in CR-induced cells, with more intracellular Tau amyloid aggregation (and with higher toxic oligomers, Fig. 6) more cellular death was observed. Upon TRZ treatment of CRinduced cells, toxicity and cell death decreased, which is likely due to prevention of the oligomerization by the drug (Fig. 7D). Comparing 7A and 7B, as observed for untreated cells, no death was observed for the cells, incubated with TRZ alone (B, TRZ alone; A, untreated). The results obtained from fluorescence microscopic imaging perfectly confirms the flow cytometry results. 3.10. Hemolytic assays One reliable/facile system for screening the toxicity of β-rich fibrils/ oligomers is the ex vivo hemolysis assay, in which the erythrocyte membranes serve as a model system for probing interactions between Tau oligomers/fibrils and the lipid bilayer. However, immature amyloid assemblies are more likely to cause membrane damage and cell death rather than mature amyloids. Evidently, Tau oligomers/fibrils demonstrated hemolytic activities against RBCs confirming their toxicity (Fig. 8, left). As expected, RBC membrane damage induced by immature amyloid assemblies (early amyloid species, t = 48 h) were higher than that of hemolytic activity of mature fibrils (late amyloid fibrils, t = 96 h). The extent of RBC damage seen with Tau fibrils was

3.11. Cytotoxicity investigation using LDH assay LDH, as a cytosolic enzyme, is released into cell culture medium following plasma membrane damage, such as during necrotic cell death. First, we observed that TRZ alone caused no significant increase in LDH release from the neuronal cells, indicating that the plasma membrane of treated cells remained almost intact upon the drug interaction. As indicated in Fig. 8 (right), formation of extracellular Tau amyloid fibrils (species) was toxic to the SH-SY5Y cells, as demonstrated by the release of lactate dehydrogenase (LDH). However, contrary to the outcome of hemolysis experiments, cell-death induced by 9

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Fig. 6. Representative fluorescence-activated cell sorting (FCAS) plots demonstrating gating in control (A), CR induced cells (B) and CR induced cells treated with TRZ (C). Mean FITC intensities (D) and Mean FITC fluorescence (E) of different treatments. For interpretation of the references to colour in this figures A–D legend, the reader is referred to the web version of this article. In (D), error bars (SD) were marginal and thus are masked by the columns. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.12. MD calculations

immature amyloid assemblies (t = 48 h) was slightly lower than that of hemolytic activity of mature Tau fibrils, withdrawn at t = 96 h. In agreement with the hemolysis assays, and as shown in Fig. 8 (right), TRZ attenuates Tau amyloid-induced cytotoxicity of the withdrawn sample at t = 96 h, but surprisingly, for Tau amyloid sample withdrawn at 48 h of incubation, the extent of toxicity seen in the presence of the drug, TRZ, at the tested concentration (50 μM), was much higher than the toxicity of (drug) untreated cells. Hemolysis results clarified that along with fibrils maturation (conversion of oligomers to matured fibrils), the cell toxicity is decreased. However, the cytotoxicity of late amyloid species (in the drug-treated samples) was slightly higher than expected. It seems that this may be due to preservation of the specific oligomeric states by the drug, since dynamic inter-conversion exists between oligomers and matured fibrils. To unravel this discrepancy, further cytotoxicity evaluations using different cell lines may be useful. Although along with fibrillation maturity, the degree of toxicity of the oligomeric states is generally reduced but accumulation of cytoplasmic aggregates within the cells can effect molecular cell trafficking and the physiology of neuronal processes [89] and causes co-assembly with the other proteins [90], including MT-associated proteins [91,92] and intracellular neurofibrillary lesions [93].

To glean further mechanistic insight into the Tau–TRZ interaction at the molecular level, molecular docking/MD approaches were applied, indicating that whether experimentally determined amyloid inhibition and/or binding data correlate with MD/docking outcomes, supporting validity of employed theoretical approaches or not. All calculations were employed on the last 80 ns of the simulation where the RMSD plot reaches a stable plateau (Fig. 9). Herein, to distinguish suitable pockets along the trajectory, the MDpocket software was used to evaluate dynamics of the putative pockets. The MDpocket analysis reveals binding sites frequently map during the simulation. Calculation of conformational sampling of the 8000 different structures along the converged system indicated that there are 8 relatively stable binding regions on Tau by the isovalue of the 0.5 to 8. The most prevalence pocket which is seen until the isovalue of the 6, located between residues of Tyr-29 and Tyr-197 (pocket 1). The next one is presented beside the residues numbering of 275–280 (pocket 2, PHF6*). Another pocket is seen near the Tyr-394 (pocket 3). These two pockets are seen during the isovalue of 0.05–2. The fourth pocket is located beside the residues of 306–310 (PHF6) at the value of 0.08–2 (pocket 4). There are 4 transient binding sites which are seen under the isovalue of 1. These are comprising of amino acids 115–123, and amino acids 135–140 (pocket 5) and beside the residues of 50–55 and 210–220 (pocket 6) (Fig. 10). Moreover, for prediction of TRZ binding site and the binding affinity on Tau, we used 10

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Fig. 7. Fluorescence microscopy images of SH-SY5Y cells untreated as control (A), TRZ alone (B), treated with CR (C) and CR + TRZ (D). In the case of live cells, AO penetrates into the cells and causes the green color fluorescence. No death was observed in all the control groups (B, TRZ alone; A, untreated). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

attenuating behavior of the drug. It is also noteworthy that thermodynamic/binding data were obtained based on Tyr fluorescence quenching behavior (Fig. 3, Table 1). The protein comprises 5 tyrosine residues in its sequence, 4 of which (Tyr-29, Tyr-197, Tyr-310 and Tyr394) are in direct contact with the drug, according to the theoretical data. To examine the other behaviors of Tau in the presence of TRZ as the ligand, a different simulation system was designed by randomly adding 10 ligands against an individual Tau. The MD simulation was organized under the same conditions as the first part and the simulation launched for the 100 ns of the timescale. The structural indicators of the RMSD, RMSF, Rg, and SASA was calculated and compared for both systems (Fig. 9). The RMSD in the simulation system of Tau ranged from 0.7 to 0.9 nm, while in the attendance of the drug diminished to 0.3–0.5 nm. The radius of gyration in the empty Tau has many fluctuations and the differences are between 2.30 and 2.60 nm, and in Tau-ligand assembly has fewer fluctuations around 2.30–2.45 nm. These data illustrated that the protein compactness is increased in the complex form. Furthermore in the SASA computation, the accessible area in the complex form was decreased which indicates increment in the protein stability. After finishing up the 100 ns of the dynamic MD simulation, the RMSF

AutoDock Vina for molecular docking which is reported as a well enough docking program in speeding up and accuracy in the prediction for the chemical compounds. Docking analysis was planned along the trajectory of the 8000 snapshots of the receptor conformation by the same ligand (TRZ). Docking extracted data showed that relatively every 20 ns, the docking scenes are different and at the last 20 ns, the features are the same as the first part. This is clearly clarified that the sampling was adequate for overcoming the structural flexibility of Tau. Docking affinities range from −7.2 to −10.3 kcal/mol. The binding region of the drug is exactly in consistent to the binding site analysis and the drug was located in 6 distinct regions including pocket 1 to 6. The frequency of the occurrence in the first 4 regions is more than the others which comprise of the (1) near the Tyr-29 and Tyr-197 (2) along the residues of 275–280 (3) near the Tyr-394 (4) along the entities of the 306–310. As indicated in Fig. 10, the fibrillation inhibitor, TRZ, (tightly) binds Tau in close proximity of the Tau amyloidogenic PHF6/PHF6* motifs, in a relatively high prevalence. Overall, The thermodynamic data (Fig. 3, Table 1), in accordance with the in silico theoretical approaches and “peptide assembly inhibition” studies (Fig. 2B), also suggest PHF6/ PHF6* segments as the main TRZ binding site on the Tau molecule, and provide a good structural basis to explain the efficient fibrillation

Fig. 8. (Left). Partial protective effect of TRZ against amyloid induced hemolysis. The hemolysis percentage of Tau amyloid aggregates [Tau samples (30 μM), incubated at 37 °C with and without TRZ (50 μM), withdrawn at two different time intervals (48 h and 96 h). No RBC hemolysis was detected for TRZ alone (50 μM) and buffer alone. B [TRZ was added to preformed Tau fibrils, incubated for 48 h]. (Right). The release of lactate dehydrogenate (LDH) from the SH-SY5Y cells, as a measure of cell death. LDH assay of Tau amyloid aggregates (with (50 μM) and without TRZ) and TRZ alone after 48 and 96 h. Three independent experiments were considered to calculate the spread in the data shown by the error bars. Some error bars (SD) were marginal and thus are masked by the figure columns. 11

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Fig. 9. The structural indicators from the MD simulation analysis. RMSD plots show that two systems are well converged from 20 to 100 ns of the time frame. The Rg plots deciphered that the Tau compactness complexed to the drug is increased and the spatial distribution of Tau atoms are dropped around the protein's center of mass. The RMSF plots illustrate that the values of the founded pockets 1 to 6 amino acids are extremely decreased. The SASA calculations indicating the presence of the ligand led to a lower exposure than the individual Tau.

descriptor showed that the fluctuation of the six binding site regions is decreased in comparison to Tau alone, since binding occurs at the same site of the docking analysis. Amino acids of the founded pockets (1–6) are involved in the binding process. These data are clearly in agreement to the docking and the binding site searching analyses. The RMSF computations also clarified that the fluctuation of the mentioned residues are extremely diminished. It is probable that formation of β-rich fibrils by the misfolded Tau molecules proceeds through a zipper-like mechanism, mediated by intermolecular Tau-Tau interactions at amyloidogenic segments [94]. From the MD results, it can be inferred that upon TRZ-Tau interaction, the protein becomes compressed, resulting in decrement in the surface contact required for interaction with other protein molecules in its vicinity, thereby reducing fibril formation. Under this condition, the two motifs, 275VQIINK280 and 306VQIVYK311, which are necessary for aggregation are buried inside the protein [51]. Of course, since these two sequences are located in the microtubule binding domain, these events may affect the interaction of Tau with microtubule. Small molecular weight compounds can also block the Tau-Tau interaction through this mechanism [95–97].

4. Conclusion Trazodone (whose primary action is a serotonin antagonist and reuptake inhibitor, and also has mild sedative effects due to its histamine receptor activity) and other licensed antidepressants, have been safely used for management of sleep problems, agitation and insomnia in AD patients, albeit usually in relatively advanced diseases. At low doses, the drug acts only via its most potent binding properties (5-HT receptors, especially 5-HT2A), but at higher doses recruits additional pharmacologic actions and becomes a “multifunctional” drug with a different mixture of pharmacologic functions [33,98,99]. Recently, several research groups have shown that TRZ reduces pTau burden, restores memory deficits, abrogates development of neurological signs, prevents neurodegeneration or markedly acts as neuroprotective in animal models of neurodegeneration [31,100–102]. La et al. suggest association between TRZ use and delayed cognitive decline in human, and highlighted their results as a reliable support for a potentially attractive and cost-effective intervention in dementia [100]. Halliday and co-workers recently showed that TRZ's antagonist activity on the 5-HT2A receptor (whose the highest affinity, Ki (Kd) = 25–35 nM 12

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Fig. 10. (Top, Middle) The MDpocket program results for putative binding sites frequently map prediction. There are six pockets characterized during the last 80 ns of the equilibrated MD simulation. The Tau is shown as colored surface representation and the pockets frequently maps are shown in yellow wireframe. In the pocket 1, Tyr-29 and Tyr-197; in the pocket 2, 275–280; in the pocket 3, Tyr-394; in the pocket 4, 306–310; in the pocket 5, 118–125; and in the pocket 6, 210–215 residues are shown as colored surface pink. (Bottom, left) Simultaneous 3D representation of TRZ (red) and pocket 2 (blue). (Bottom, right) Simultaneous 2D representation of TRZ (CPK) and pocket 2 (red). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.).

Table 1, it is estimated that Kd values of the drug for binding to 1N4R Tau, in vitro, are ~2000–10,000 nM at different temperatures. Regarding high levels of 1N4R Tau isoform in the brain [104], significant intracellular and cerebrospinal fluid concentrations of Tau (300–400 pg/ml; and elevated levels of Tau in AD patients as well [105,106] and considering competitor receptors for TRZ binding, there is still the strong possibility that the “neuron-penetrating TRZ molecules” bind to the monomeric/oligomeric forms of Tau, in vivo and affect pathologic Tau fibrillar assembly. Structurally, also, it is not so strange that TRZ is capable of affecting Tau oligomerization and amyloid fibrillation of the protein as well. The backbone hydrogen bonding and hydrophobic interactions of Tau amyloidogenic segments (PHF6/PHF6*) are suggested as main contributors in stabilizing the fibrils. Since TRZ possesses ideal structure features, including a flexible backbone, hydrophobic nature, and several available hydrogen bond (H-bond) acceptors, it may interact within hydrogen bonded strands (or mask amyloidogenic regions) and disrupt the hydrophobic interactions via intercalating between stacked

for TRZ [99,103]) decrease p38 activity leading to reduced levels of activating transcription factor 4 (ATF-4) (a key mechanism in slowing neurodegeneration) and proposed a possible beneficial use of trazodone in dementia and AD [31]. As secondary mechanism of action, TRZ as 5HT1A agonist, is expected to lead to ERK1/2 activation and increased expression of brain-derived neurotrophic factor as well [35,99]. In the current study, we propose a different mechanism of TRZ action that exerts beneficial effects on dementia and AD. We found that this drug affect Tau fibrillation and also is capable of attenuating of the production of toxic oligomeric species. Prerequisite for this activity is the tight binding of TRZ to the protein. A typical hypnotic dose of TRZ is 50 mg at night which affords, after approximately 1 h following oral administration, a Cmax in the brain of ~0.4 μg/ml (~1 μM/1000 nM). At this (drug) concentration, the predicted occupancy for high affinity receptors; “5-HT1A (Ki = ~120 nM), 5-HT2A (Ki = ~35 nM)” and low affinity receptors; “5-HT1B (Ki > 10,000 nM) and 5-HT1E (Ki > 10,000 nM) receptors” are expected to be more than 90 and ~10% occupied, respectively [35,99]. However, regarding Kb values in 13

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aromatic rings of these amyloidogenic segments. So, the drug intervention at both early and later stages of Tau fibrillation process is likely (Scheme 1). Finally, TRZ appears to have potential to be considered for ADmodifying treatment. Thus, there is a possibility that researchers consider re-evaluating the drug in future clinical investigations on AD.

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Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Acknowledgments The author (R. K.) received financial supports from National Institute for Medical Research Development (NIMAD, grant No. 984494) and research Council of Kermanshah University of Medical Sciences (Grant No. 980308) for the research, authorship, and/or publication of this article. Also, R.K. kindly acknowledge Dr. Markus Zweckstetter as well as Shaolong Zhu and Dr. Derek J. Wilson for providing the PDB coordinate file for the ‘native’ Tau ensemble predicted from their NMR/HDX work and are also very grateful to Dr. J. Avila (Universidad Autonoma de Madrid, Spain) for Tau (1N4R) plasmid. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.abb.2019.108218. References [1] J. Cummings, P.S. Aisen, B. DuBois, L. Frölich, C.R. Jack, R.W. Jones, J.C. Morris, J. Raskin, S.A. Dowsett, P. Scheltens, Drug development in Alzheimer's disease: the path to 2025, Alzheimer's Res. Ther. 8 (1) (2016) 39. [2] B. Dubois, H.H. Feldman, C. Jacova, H. Hampel, J.L. Molinuevo, K. Blennow, S.T. DeKosky, S. Gauthier, D. Selkoe, R. Bateman, Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria, Lancet Neurol. 13 (6) (2014) 614–629. [3] A. Takashima, Tau aggregation is a therapeutic target for Alzheimer's disease, Curr. Alzheimer Res. 7 (8) (2010) 665–669. [4] E.D. Roberson, K. Scearce-Levie, J.J. Palop, F. Yan, I.H. Cheng, T. Wu, H. Gerstein, G.-Q. Yu, L. Mucke, Reducing endogenous Tau ameliorates amyloid ß-induced deficits in an Alzheimer's disease mouse model, Science 316 (5825) (2007) 750–754. [5] M.D. Weingarten, A.H. Lockwood, S.-Y. Hwo, M.W. Kirschner, A protein factor essential for microtubule assembly, Proc. Natl. Acad. Sci. 72 (5) (1975) 1858–1862. [6] O. Schweers, E. Schönbrunn-Hanebeck, A. Marx, E. Mandelkow, Structural studies of Tau protein and Alzheimer paired helical filaments show no evidence for betastructure, J. Biol. Chem. 269 (39) (1994) 24290–24297. [7] D.W. Cleveland, S.-Y. Hwo, M.W. Kirschner, Physical and chemical properties of purified Tau factor and the role of Tau in microtubule assembly, J. Mol. Biol. 116 (2) (1977) 227–247. [8] M.-K. Cho, H.-Y. Kim, P. Bernado, C.O. Fernandez, M. Blackledge, M. Zweckstetter, Amino acid bulkiness defines the local conformations and dynamics of natively unfolded α-synuclein and Tau, J. Am. Chem. Soc. 129 (11) (2007) 3032–3033. [9] V.N. Uversky, Natively unfolded proteins: a point where biology waits for physics, Protein Sci. 11 (4) (2002) 739–756. [10] F. Avbelj, R.L. Baldwin, Role of backbone solvation and electrostatics in generating preferred peptide backbone conformations: distributions of phi, Proc. Natl. Acad. Sci. 100 (10) (2003) 5742–5747. [11] D.A. Brant, P.J. Flory, The configuration of random polypeptide chains. II. Theory, J. Am. Chem. Soc. 87 (13) (1965) 2791–2800. [12] C. Tanford, K. Kawahara, S. Lapanje, Proteins in 6 M guanidine hydrochloride demonstration of random coil behavior, J. Biol. Chem. 241 (8) (1966) 1921–1923. [13] S. Jeganathan, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, The natively unfolded character of Tau and its aggregation to Alzheimer-like paired helical filaments, Biochemistry 47 (40) (2008) 10526–10539. [14] Z.A. Levine, L. Larini, N.E. LaPointe, S.C. Feinstein, J.-E. Shea, Regulation and aggregation of intrinsically disordered peptides, Proc. Natl. Acad. Sci. 112 (9) (2015) 2758–2763. [15] S. Müller-Späth, A. Soranno, V. Hirschfeld, H. Hofmann, S. Rüegger, L. Reymond, D. Nettels, B. Schuler, Charge interactions can dominate the dimensions of intrinsically disordered proteins, Proc. Natl. Acad. Sci. 107 (33) (2010) 14609–14614.

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