The effect of Benzothiazolone‐2 on the expression of Metallothionein‐3 in modulating Alzheimer's disease

Abstract Introduction Metallothioneins (MTs) are a class of ubiquitously occurring low‐molecular‐weight cysteine‐ and metal‐rich proteins containing sulfur‐based metal clusters. MT‐3 exhibits neuro‐inhibitory activity. The possibility to enhance the expression of MT‐3 or protect it from degradation is an attractive therapeutic target, because low levels of MT‐3 were found in brains of Alzheimer's disease (AD) patients. Objectives The primary objective of this study was to test an enhancement of MT‐3 cellular concentration after MT‐3 binding treatment, which could prevent MT‐3 degradation. Methods MTT assay, flow‐cytometry, fluorescence microscopy, quantitative real‐time polymerase chain reaction, and immunodetection of MT3 were used for analysis of effect of STOCK1N‐26544, STOCK1N‐26929, and STOCK1N‐72593 on immortalized human microglia‐SV40 cell line. Results All three tested compounds enhanced concentration of MT‐3 protein in cells and surprisingly also mRNA concentration. IC50 values of tested molecules exceeded about ten times the concentration that was needed for induction of MT‐3 expression. The tested compound Benzothiazolone‐2 enhanced apoptosis and necrosis, but it was not of severe effect. About 80% of cells were still viable. There was no serious ROS‐generation and no severe decrease in mitochondria numbers or stress induced endoplasmic reticulum changes after test treatments. The selected compound showed stable hydrophobic and electrostatic interaction during MT‐3 ligand interaction. Conclusion Benzothiazolone‐2 compounds significantly enhanced MT‐3 protein and mRNA levels. The compounds can be looked upon as one of the probable lead compounds for future drug designing experiments in the treatment of Alzheimer's disease.


| INTRODUCTION
Alzheimer's is the most common form of dementia; it is a term for memory damage and other intellectual capacities that are serious enough to impede daily life. Alzheimer's disease accounts for 60 to 80 percent of dementia cases. The neuropathological hallmarks of Alzheimer disease (AD) are comprised of "positive" lesions such as amyloid plaques, cerebral amyloid angiopathy, neurofibrillary kinks, glial responses and "negative" lesions such as neuronal and synaptic loss (Haass & Selkoe, 2007;Iqbal, Liu, Gong, Alonso Adel, & Grundke-Iqbal, 2009). The neurochemical elements responsible for this age-linked compulsive advance are still poorly described. The growing evidence supports a significant role for biometals such as copper (Cu), iron (Fe), and zinc (Zn) in Aβ accretion and neuronal relapse (Bush & Tanzi, 2008;Lovell, Robertson, Teesdale, Campbell, & Markesbery, 1998;Miu & Benga, 2006;Roberts, Ryan, Bush, Masters, & Duce, 2012;Zatta, 2008). The plasma Cu/MT ratio was found to be symptomatic of disease advancement in AD patients. This connotation hints at a probable MT-4 is present in squamous epithelial tissue (Palacios, Atrian, & Capdevila, 2011).
Degradation and deficiency in MT-3 may lead to severe disruption of lysosomal biogenesis and cytoskeleton dynamics. Dysfunction of lysosomes and actin cytoskeleton in MT-3-null astrocytes contributed to accumulation of toxic Aβs proteins and other damaged proteins.
Apart from the function of MT-3 in lysosomes and cytoskeleton, homeostasis of metal ions managed by MT-3 may also play a key role in AD (Bonda et al., 2011). Furthermore, MT-3-null astrocytes had low level of zinc in lysosome and presented autophagy defect. Autophagy seems to be abnormal due to alteration of the endo-lysosomal pathway, which impairs fusion of autophagosomes with lysosomes. In addition, a recent report details the contribution of mitophagy, a specialized form of autophagy that removes damaged mitochondria in AD (Moreira et al., 2007).
Interestingly, the activation of autophagy can promote degradation of APP/Aβ and reduce tau pathology (Nixon, 2007). Therefore, the autophagy pathway is considerably more complex in AD because it is simultaneously induced and impaired. APP/Aβ can be generated in autophagosomes at low levels in a physiological environment; however, during disease conditions autophagosomes rapidly accumulate in the cell body and axonal terminals due to either impaired autophagic flux or a defected endosomal pathway (Lee, 2009;Lee, Park, Kim, & Koh, 2010). Martin et al. (2006) showed that in the Tg2576 transgenic mouse model of AD, MT-3 was less than that found in wild-type mice. It also establishes the roles of MT-3 and Nnos in Alzheimer's disease. The pathological changes that develop in this mode might be responsible for the degradation of MT-3. Roy, Kumar, Baig, Masarik, and Provaznik (2015) reported that natural-based compounds were effective in modulating Alzheimer's disease. The in silico findings (which include studies of both pharmacodynamics and pharmacokinetic properties) show that MT-3 was able to bind with STOCK1N-26544, 26929, and 72593 compounds.
The former two compounds contain Benzothiazolone-2 as a frag-

| Cell cultures and cultured cell conditions
The immortalized human microglia-SV40 (Applied Biological Materials Inc., Richmond, BC, Canada) were used in this study. These cells are positive for NGF [nerve growth factor (Beta Polypeptide)], Iba1 ionized calcium-binding adapter molecule 1, TREM2 (triggering receptor expressed on myeloid cells 2), CD11b, and CD68. The immortalized human microglia was cultured in Prigrow III Medium with 10% fetal bovine serum in collagen-coated flasks. The medium was supplemented with penicillin (100 U/ml) and the cells were maintained at 37°C in a humidified incubator with 5% CO 2 .

| RNA isolation and reverse transcription
TriPure Isolation Reagent (Roche, Basel, Switzerland) was used for RNA isolation. RNA samples without reverse transcription were used as the negative control for qRT-PCR, in order to exclude DNA contamination. The isolated RNA was used for the cDNA synthesis. RNA (1000 ng) was transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche), which was applied according to the manufacturer's instructions. The cDNA (20 μl) prepared from the total RNA was diluted with RNase-free water to 100 μl; 5 μl was then directly analyzed using the LightCycler ® 480 II System (Roche).

| Quantitative real-time polymerase chain reaction
qRT-PCR was performed using TaqMan gene expression assays and the LightCycler ® 480 II System (Roche). The amplified DNA was analyzed by the comparative Ct method using β-actin as a reference gene. The primer and probe sets for ACTB (assay ID: Hs99999903_m1) and MT-3 (assay ID: Hs01921768_s1) were selected from the TaqMan gene expression assays (Life Technologies, USA). The qRT-PCR was performed under the following amplification conditions: total volume of 20 μl, initial incubation at 50°C/2 min followed by denaturation at 95°C/10 min; then 45 cycles at 95°C/15 s and at 60°C/1 min. All samples were measured in duplicates.

| Immunodetection of MT-3
Extraction of total protein from the cells was processed using the Pierce ® RIPA Buffer (Thermo Scientific, Rockford, IL, USA) in accordance with the protocol provided. All protein concentrations were determined with the Pierce ® BCA Protein Assay Kit. Protein samples were diluted to 10 μg/μl, then 1 μl was slowly spotted onto a 0.2μmImmun-Blot ® PVDF membrane (BioRad Laboratories, Hercules, CA, USA) and air-dried. To block nonspecific binding sites, the membranes were incubated using 5% Blotting-Grade Blocker (BioRad Laboratories) for one hour at room temperature. They were then incubated using Anti-MT-3 antibody (product no. ab76618; Abcam, Cambridge, UK), and with Antiβ-actin (product no. ab8227; Abcam).
The membranes were then washed four times in PBS (Sigma-Aldrich, St. Louis, MO, USA) for five minutes each, before being incubated with a secondary antibody (Peroxidase-Labeled Anti-Rabbit IgG (product no. PI-1000; Vector, Burlingame, CA, USA)). This was diluted 1:2000 using 5% Blotting-Grade Blocker. After washing in PBS, all blots were visualized on photosensitive film. Densitometric analyses of the membranes were performed using GeneSnap (Syngene, Cambridge, UK).

| Cytotoxicity testing -MTT test
The suspension of 5000 cells was added to each well of standard microtiter plates. A volume of 200 μl was transferred to wells 2-11. The medium (200 μl) was added to the first and the last column (1 and 12(control)). The plates were incubated for 2 days at 37°C to ensure cell growth. The medium was removed from columns 2-11. Columns 3-10 were filled with 200 μl of the medium, which contained an increased concentration of tested compound diluted in dimethylsulfoxide (DMSO) (0-1 μmol/l). As a control, columns 2 and 11 were filled with the medium without tested compound.
The plates were incubated for 12 and 24 hr, then the medium was removed and the cells were washed in PBS. Columns 1-11 were filled with 200 μl of medium, which contained 50 μl of MTT (5 mg/ ml in PBS) incubated in a humidified atmosphere for four hours at 37°C and then wrapped in aluminum foil. After the incubation, the MTT-containing medium was replaced with 200 μl of 99.9% DMSO in order to dissolve the MTT-formazan crystals. Subsequently, 25 μl of glycine buffer was added to all wells; absorbance was immediately determined to be 570 nm (VersaMax microplate reader, Molecular Devices, Sunnyvale, CA, USA). All samples were measured in duplicates.

| Confocal microscopy and cell staining
For the purpose of fluorescence microscopy, the cells were cultivated directly on microscope glass slides (75 × 25 mm, thickness 1 mm, MenzelGlässer, Braunschweig, Germany) in Petri dishes in the abovedescribed cultivation media (see "Cultured cell conditions"). The cells were transferred directly onto the slides, which were then submerged in the cultivation media.
After the treatment, the microscope glass slides with a monolayer of cells were removed from the Petri dishes, rinsed in the cultivation medium without supplementation and PBS buffer and directly used for staining and fluorescence microscopy. The cells were incubated using the following highly specific fluorescent probes:

| Thermodynamic studies -MD simulation
The MM-PBSA (Molecular Mechanics energies combined with Poisson-Boltzmann Surface Area) approach, combined with molecular dynamics (MD) simulations, is widely used in biomolecular complexes as a way to estimate the protein ligand interaction energies and predict binding-free energies. It also evaluates the relative stabilities of different biomolecular structures. Additionally, it can be used to rescore a set of docked complexes, thereby improving the ability to distinguish between active and inactive lead molecules. The molecular dynamics simulation of MT-3 docked complexes was performed using the GROMACS 4.5.52 (Berendsen et al., 1995;Lindahl et al., 2001) package, with a standard GROMOS96 force field for 10,000 ps.
Binding free-energy calculations were performed in this work for the ligand-bound complexes in order to provide further insight into the study.

| The cytotoxic evaluation of benzothiazolone-2containing substances in microglia cells
The IC50  The AnnexinV+/PI+ (Q2 quadrant) depicts late necrosis and dying cells. The rate of the necrosis triggered by tested compounds did not exceed 4% (only showing a 2.12% increase, compared to the stained control). The Annexin V+/PI-(Q4) population represents apoptotic cells or early oncotic cells (see Figure 2). The largest amount of apoptotic or early oncotic cells was found in 10 nmol/L STOCK1N-72593 after treatment (19.93%; 12.31% increase compared to the stained control). Nevertheless, some portion of oncotic cells is able to reverse oncosis and could thereby survive; thus, no severe increase in the total number of dying cells was found after treatment with tested compounds (see Figure 2).
Confocal microscopy revealed no significant morphological changes that had been induced by studied compounds. On the other hand, we used two probes to localize mitochondria and the endoplasmic reticulum. Whereas MitoTracker Green FM localizes mitochondria regardless of mitochondrial membrane potential, ER-Tracker Red, a conjugate of green-fluorescent BODIPY ® TR dye and glibenclamide, also reflects expression of sulphonylurea receptors of ATP-sensitive K + channels and thereby reflects the ER function. The double-staining revealed no significant decrease in fluorescence corresponded to both mitochondria and ER for the tested compounds. Compound 26544 showed some ability to generate ROS, which were colocalized with mitochondria (see Figure 3). Nevertheless, ROS production was rather weak.

| Benzothiazolone-2 enhances expression of MT-3 in microglia cells
mRNA expression of MT-3 was relatively low in control cells without treatment. All the three tested compounds enhanced expression of MT-3 mRNA in both tested concentrations (5 nmol/L and 10 nmol/L, respectively) (see Figure 4a).

| Thermodynamic studies on MT-3-benzothiazolone complexes
Binding free-energy studies that validate the above findings were also performed. The analysis was carried out for the Benzothiazolone-2 docked complexes only, since the above results were encouraging. The molecular docking representation is provided in Figure S1.
It clearly states that the tested molecules (STOCK1N-26929 and STOCK1N-26544) both have stable binding free-energies values in the form of potential, polar, and nonpolar solvation energies that are comparable to the standard controls (Table 1). It also states that both hydrophobic and electrostatic interactions are important for protein ligand interaction in case of MT-3 for Alzheimer's disease.

| DISCUSSION
The possibility to enhance the expression of MT-3 or protect it from degradation is an attractive therapeutic target, because low levels of MT-3 were found in AD brains. In this study, we tested an The expression of MT-3 protein was undetectable in control cells without treatment. All three tested compounds enhanced concentration of MT-3 protein in cells and surprisingly also mRNA concentration. Nevertheless, STOCK1N-72593 was relatively less effective. It is to be noted here that different concentrations of tested compounds can influence expression of MT-3 differently. Epigenetic mechanisms and different types of activation of signaling pathways are suspected in this case. Furthermore, the expression of MT-3 could be regulated at the posttranscriptional level, which was also observed in earlier studies (Garrett et al., 2005). The experiments carried out did not assume that triggering of MT-3 expression should be in positive correlation with tested compound concentration. The results showed that our compounds are able to influence MT-3 expression. It is to be mention that our study is not oriented toward exact mechanism or to show exact quantities of MT-3 protein. In our opinion, two of our compounds significantly enhanced MT-3 protein and mRNA levels.
According to fluorescence staining, no serious ROS generation and no severe decrease in mitochondria numbers or endoplasmic reticulum changes after test-treatment were found. This is very important, (because oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress have been implicated in beta-amyloid neurotoxicity).
The above findings were further validated by calculating the bind-  binding interactions is governed by the hydrophobic forces. The second major component was electrostatic energy, which is comparably higher in STOCK1N molecules than in controls.
The overall findings suggest that hydrophobic and electrostatic interaction is imperative for current MT-3 ligand interaction. Polar solvation and nonpolar solvation energies are comparable to the standard controls (Table 1).

| CONCLUSIONS
The current work has made an effort to see the effect of Benzothiazolone-2 on the cellular concentration of MT-3. Many studies show that MT-3 mRNA is downregulated in AD brains and that this might, therefore, contribute to the upsurge of abnormal neuronal development associated with the disease.
Our previous findings suggests that Benzothiazolone-2 possesses better pharmacokinetic and pharmacodynamics properties.
It also forms stable complex with MT-3 according to molecular dynamics findings. The above findings were validated by screening the compounds against live immortalized human microglia cells (SV40).
This tested compound showed neither severed cytotoxicity nor did it trigger necrosis. In addition, there was no serious ROS-generation, decrease in mitochondrial numbers, stress-induced endoplasmic reticulum changes after test-treatments. All three tested compounds en-