DIZE was purchased from Santa-Cruz Biotechnology (cat no. sc-205651) and freshly prepared daily. STZ was procured from Calbiochem (catalog no. 572201). All other rats received equivalent volumes of vehicle (saline) injections.
A competitive ACE2 antagonist, MLN4760 was procured from Merck Millipore (CAS 305335-31-3). It shows high binding affinity towards ACE2 enzymatic active site and significantly alters the ACE2 protein conformation . The MLN4760 was administered concomitantly with DIZE using a similar vehicle, at 10µM concentration. This dose was selected on the basis of previously done studies using MLN-4760 studies [39, 38]. All other chemicals and reagents were of analytical grade.
2.1. Cell culture and treatment
Mouse neuroblastoma cell line (Neuro-2A/N2A cell) was procured from National Centre for Cell Sciences, Pune, India, and supplemented with DMEM encompassing 10% FBS at 37℃ in a controlled atmosphere (5% CO2/95% air). STZ was freshly prepared in serum free media at the time of administration in serum-free media. N2A cells were incubated with STZ (2.5 mM, optimized using MTT assay; Supplementary Fig. 1A and B ) [40, 41] in absence or presence of DIZE (25 µM, lowest effect dose standardized using MTT assay; Supplementary Fig. 1C and D) and DIZE alone for 24 h. Cells were treated with DIZE after 30 min of STZ addition.
2.1.1. Primary Astrocyte culture
Astrocyte culture media (30 ml) was pre-warmed (DMEM F-12 + 10% heat-inactivated fetal bovine serum + 1% Penicillin/Streptomycin) to 37 0C. T-75 flask was coated with 10 ml of poly-D-lysine (PDL) (50 µg/ml) dissolved in cell culture grade water and placed at 37 0C in CO2 incubator for 2 hr. For the dissection procedure, HBSS media was prepared and pH was adjusted between 7.2 to 7.4. Pups were sacrificed following the decapitation process and brain was isolated and minced under sterile conditions with the addition of HBSS buffer. After removing HBSS buffer, and 2.5% trypsin was added for tissue dissociation and placed in incubator at 37 0C for 30 min. The mixture was centrifuged (5 min at 300 x g) and the pellet was carefully collected. Tissue was triturated in astrocyte plating medium (10 ml) with the help of a 1 ml and 10 µl pipette. The pellet was passed through a 70-micron filter to obtain single-cell suspension and the cells were subsequently plated in a flask. After achieving approximately 70-80% confluece the flasks were placed on an orbital shaker (180 rpm for 30 min) to remove the supernatant containing microglia. To further remove oligodendrocyte precursor cells (OPC), fresh medium (20 ml) was added to the astrocyte culture and flask remained in shaking condition at 240 rpm for 6 hr. Finally, the confluent astrocyte cell layer was washed twice with PBS and incubated with fresh medium in CO2 incubator at 37 0C for further experimentation.
2.1.2. Estimation of Ang-(1-7) level
The Ang (1-7) level was estimated in isolated brain tissue of treated and untreated rats/N2A cells using conventional ELISA kit (Fine Test, China) according to the manufacturer’s protocol. In brief, the evacuated brain tissue of treated and untreated rats/harvested N2A cells were homogenized in ice-cold PBS with 5% (w/v) protease inhibitor cocktail (Sigma) followed by sonication (10 secs; 20 cycles) using an ultrasonic processor (Heat Systems-Ultrasonic inc.) and centrifugation at 20,000 g (15 min) at 4 0C. The data was expressed in the terms of pg/ml and pg/mg for culture supernatants and brain tissue, respectively .
2..1.3. Oxidative stress marker
22.214.171.124. Estimation of mitochondrial ROS
Mitochondrial ROS was estimated using Mito SOX Red (Invitrogen, USA), which is a live-cell permeant that rapidly and selectively targets mitochondria . Once in the mitochondria, Mito SOX Red reagent gets oxidized by superoxide and imparts red colored fluorescence (with excitation at 510 nm and emission at 580 nm). N2A cells were seeded in 6-well plates (2 × 105 cells/well), and treated with STZ (2.5 mM), with or without DIZE for 24 h. After 24h of treatment cells were incubated with 5 µM Mito SOX Red for 10 min at 37 0C, and washed twice with PBS., Fluorescence signal was detected using a laser system (FACS calibur; BD bioscience, USA) with 510 nm excitation and 580 nm emission filters.
126.96.36.199. Estimation of reduced Glutathione (GSH) level
Reduced glutathione was estimated by 5, 5-dithiobis (2-nitrobenzoicacid) [DTNB, Ellman's reagent that yields a yellow colored product. N2A cells were plated (4X105 cells/well). Following treatment, cells were scraped by adding 100 µl of PBS (ice cold). Cells were lysed by ultra-sonication, and centrifuged at 10,000 g for 5 min at 4 0C. Subsequently 10% trichloroacetic acid (100 µl) was added to this lysate for protein precipitation and suspension was kept on ice for 1 hr. Samples were re-centrifuged at 5000 g for 5 min and supernatant was collected. Further supernatants (75 µl) was mixed with 25 µl of distilled water followed by 100 µl of buffer and DTNB (50 µl). Reaction mixture was incubated for 10 min and read at 412 nm. .
2.1.4. Acetylcholinesterase Activity Estimation
AChE activity was estimated by the method described by Ellman et al.  with a few minor changes. After treatment, the cells were scraped and lysed in 0.1 M sodium phosphate buffer (PB, pH 8.0) with Triton-X 100 (0.1 %) followed by centrifugation at 10,000× rpm for 20 min at 4 0C subsequently supernatant was mixed with 100 µM of 5,5′-dithio-bis (2-nitrobenzoic acid (DTNB) and 0.1 M PB. After 10 min incubation at 37 0C, 20 mM acetylthiocholine iodide was added to initiate the enzymatic reaction. The enzymatic activity was measured at 412 nm for 2 min at a 15 sec interval by spectrophotometer and specific activity of AChE was presented in µmoles / min / mg of protein.
2.1.5. Intracellular Ca2+ level estimation
Intracellular Ca2+ level was studied using the manufacturer's protocol. In brief, N2A cells (5x104) were firstly incubated with 5 µM Fluo4-AM at 37°C for 30 min in dark. Cells were washed with PBS to remove the extracellular Fluo4-AM and resuspended in PBS. The fluorescence signal was detected using a laser system (FACS calibur; BD bioscience, USA). An argon laser was used to excite Fluo4 at 494 nm, and the emission was measured at 506 nm. The fluorescence intensity for each sample was determined by using Cell Quest software (BD bioscience, USA)
2.1.6. Mitochondrial membrane potential (MMP/ΔΨm) measurement
Mitochondrial membrane potential (MMP/ΔΨm) was assessed using mitochondrial membrane potential sensitive carbocyanine dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzamidazolocarbocyanine iodide, Sigma Aldrich, USA) in N2A cells [45, 46]. After treatment cells were incubated with JC-1 dye (2.5 µg/ml, Sigma Aldrich, USA) in dark for 30 min at 37 0C. After incubation, the cells were washed with PBS and Fluorescence signal was detected using a laser system (FACS calibur; BD bioscience, USA) with 488 nm excitation and 590 nm emission filters .
2.1.7. Western blot analysis
Freshly extracted treated cells and brain tissues were lysed using cold RIPA lysis buffer in the presence of 1X protein inhibitor cocktail (Sigma, USA) and dithiothreitol (DTT, 1mM) as per manufacturer’s instruction to extract protein. [48, 49]. Protein was separated on 10-15% SDS-PAGE subsequently transferred onto the PVDF membrane (Millipore, Bedford, MA, USA). The membrane was probed with bovine serum albumin (BSA, 5%) for 2 hr at room temperature and subsequently incubated overnight at 4 0C with primary antibodies; rabbit anti-ACE2 (1:1000, catalogue no.: AF5165; Affinity biotech Cincinnati, USA), rabbit anti-MasR (1:500, catalogue no. NBP1-78444; Thermo fisher scientific, USA), rabbit anti-NFκB (1:500, catalogue no.: sc-109; Santa Cruz Biotechnology CA, USA), rabbit anti- NLRP3 (1:1000, catalogue no. DF7438; Affinity biotech Cincinnati, USA), rabbit anti-caspae-1 (1:1000, catalogue no. AB1871; Sigma Aldrich, USA), rabbit anti-IL-1β (1:1000, catalogue no. PA5-95455;Thermo fisher scientific, USA), mouse anti-Aβ42 (1:1000, catalogue no. NBP2-13075; Novus Biologicals, LLC, USA), rabbit anti p- TAU (1:1000, catalogue no. NBP2-20574; Novus Biologicals, LLC, USA), rabbit anti-TAU (1:500, catalogue no. sc-32274; Santa Cruz Biotechnology CA, USA), mouse anti- β-actin (1:2000, catalogue no. A5316; Sigma Aldrich, USA). Following the incubation, the membrane was washed with washing buffer (pH 7.6, TBS, 0.1% Tween 20) and probed with horseradish peroxidase (HRP) conjugated secondary antibody (anti-mouse IgG/anti-rabbit IgG, 1:5000, GeNei, India) for 2 h at room temperature. Signal intensity was estimated by using ECL (enhanced chemiluminescence) Millipore MA, USA) and quantified by myImage analysis software (Thermo Scientific, USA).
2.1.8. Immunocytochemistry (ICC)
ICC study was performed as per the protocol described earlier with minor modification . Cells were incubated with primary antibodies; rabbit anti-ACE2 (1:1000, catalogue no.: AF5165; Cincinnati, USA), rabbit anti-MasR (1:500, catalogue no. NBP1-78444; Thermo fisher scientific, USA), rabbit anti-NFκB (1:200, catalogue no.: sc-109; Santa Cruz Biotechnology CA, USA), mouse anti-GFAP (1:500, catalogue no.: MAB360; Millipore, MA, USA) were incubated for 8-12 hr at 4 0C. Subsequently the cells were kept with respective Alexa Fluor-conjugated secondary antibodies (1:400) for 1 hr. After washing with PBS, cells were mounted firmly on slides followed by counterstaining with DAPI (Sigma Aldrich, USA). Images were taken under Leica inverted fluorescent microscope (DMI 6000) equipped with a digital CCD camera (Leica, Wetzlar, Germany) and quantified by ImageJ software (NIH, USA).
Adult male Sprague-Dawley (SD) rats (180-200 g) were obtained from National Laboratory Animal Centre (NLAC) of Central Drug Research Institute, Lucknow India. It is well documented that estrogens impart many beneficial effects in the brain including enhanced neuronal viability and reduced Aβ accumulation. It is also reported that mitochondria from young females are less vulnerable for Aβ toxicity, generate lesser ROS, as well as release of lower apoptotic signals compared to those of males [51, 52]. Therefore, the male SD rats were the choice of animals in the present investigation targeting STZ induced. Animals were kept in a pathogen-free cages (dimension 435 x 290 x 150 mm) in groups of n=3 per cage and controlled environment condition (23-25 0C and 60-65% humidity), under 12 hr light/dark cycles along with free access to food (normal chow diet) and water. Experimental procedures were approved by institutional animal ethics committee and met the guidelines of Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA); (approval no. (IAEC/2019/55/REN).
2.2.1. Streptozotocin administration and DIZE treatment
188.8.131.52. Stereotaxic injection of streptozotocin
SD rats were anesthetized by intraperitoneal administration of pentobarbitone sodium (40 mg/kg, Sigma-Aldrich USA). Rats were carefully mounted on stereotaxic apparatus (Stoelting Co. USA) and their ears were fixed with the ear bars in the apparatus frame. The skin on the skull was sterilized and a longitudinal cut was precisely made to expose the skull, followed by careful wiping for bregma visualization. Two small burr holes were made in skull laterally and STZ (10 µl of 1 mg/kg in artificial cerebrospinal fluid (aCSF) was infused in each lateral ventricles at following coordinates: -0.8 mm anteroposterior (AP), ±1.5 mediolateral (ML), -3.6mm dorsoventrally (DV) measured from bregma (Hamilton company, Switzerland) at a flow rate of 0.5 µl/min [53–55]. Lingered the syringe at aforementioned place for some time to reduce seepage. Likewise, the control group was administered same amount of aCSF in each ventricle.
184.108.40.206. DIZE treatment
Animals were randomly divided into 5 groups with 6 rats in each group. Group I that served as control was stereotaxically administered with 10 µl of aCSF in each lateral ventricle (Box:1). The rats of group II (Neurotoxin) were stereotaxically administered with 10 µl of STZ (1 mg/kg) in each ventricle. Group III and IV were together considered as the treatment group in which rats were treated with different doses of DIZE (10 mg/kg and 15 mg/kg, i.p.) respectively 3-day post STZ administration for 21 days [16, 56–58]. The group V was treated with 15 mg/kg (i.p.) of DIZE for 21 days and served as per se group. Doses of 10 and 15 mg/kg were equally effective in improving memory functions (refer to section 3.2) with no effect on hemodynamic parameters (Supplementary Fig. 3). For subsequent in vivo experimentation, a minimum effective dose (10 mg/kg, i.p.) of DIZE was used.
2.2.2. Behavioral parameter
220.127.116.11. Morris water maze test
Morris water maze test was performed to assess the spatial learning and memory after DIZE treatment according to a previously published protocol [59, 54, 55]. The test was executed using a round pool (132 cm ⅹ 75cm) having a platform submerged below the water meniscus and maintained at a temperature of 25±0.5°C. Spatial and learning memory performance was conducted on day 18-20 post DIZE treatment accompanied by probe trial on day 21. On day 1 rats were allowed to swim in pool and the trial ended either by animal finds the platform or trial ends (2 min). Similarly, trials were conducted on day 2 and day 3 for proper acquisition of memory, and escape latency was calculated. On day 21 Probe trial was done in which the platform was removed and animal was allowed to swim for 1 minute. Data was recorded and analyzed using Anymaze software.
2.2.3. Acetylcholine level estimation
Acetylcholine level in brain tissue of treated and untreated rats was measured by using LC-MS/MS (ABSciex 4000, Toronto, Canada) equipped with an API electro-spray ionization (ESI) source. The instrument and compound parameters were optimized and set as follows: curtain gas, auxillary gas, and collision gas, were set at 30, 35, and 30 and declustering potential (DP), collision energy (CE), entrance potential (EP), and collision exit potential (CXP) were set at 135, 12, 10 and 30 V. respectively. The ion spray voltage was set at 5500V and zero air was used as source gas whilst the nitrogen was used as both curtain and collision gas. The mass spectrometer was operated at ESI positive ion mode and the ions were detected in the multiple reaction monitoring (MRM) mode, monitoring the transition of m/z 146.20 precursor ion [M + H]+ to the m/z 87.10. Separation was done through phenomenex Luna HILIC (3 µm, 150 x 4.6 mm) column with a mobile phase consisting of acidified acetonitrile: 0.1% formic acid in the ratio of 90:10 (v/v) at a flow rate of 0.5 ml/min. Data acquisition and quantitation were performed using analyst software version 1.4.1 (Applied Biosystems, MDS Sciex Toronto, Canada).
For extraction of acetylcholine from brain tissue, a simple protein precipitation technique was followed as described in previous literature . In brief, cold acetonitrile (extracting solvent) was added to the homogenized brain tissue (3:1). The mixture was vortexed (10 min) and further centrifuged at 10,000 rpm for 10 min. Supernatant was separated and injected into column for LC-MS/MS analysis.
Rats were euthanized using high dose of sodium pentobarbital (50 mg/kg, i.p.) followed by perfusion with ice-cold paraformaldehyde (PFA, 4%). The whole brain was fixed by preserving in PFA overnight at 4 0C and dehydrated serially with varying concentrations of sucrose (10%, 20%, and 30%) for 24 hr each. Free-floating slices across the cortex and hippocampus (30 µm thick) were treated with primary antibody, mouse anti-GFAP (1:500, Millipore, CA, USA)as mentioned in our prior publication . Slices were firmly fixed on slides and counterstained with DAPI (Sigma Aldrich, USA). Fluorescent images were collected using Leica inverted fluorescence microscope (DMI 6000, Leica, Wetzlar, Germany) and quantified by ImageJ software (NIH, USA).
2.4. Statistical Analysis
All data were expressed as mean±SEM and statistical analysis was done by using Graph Pad Prism software (San Diego, CA, USA) by applying One way/two-way analysis of variance (ANOVA) followed by bonferroni post hoc test and P < 0.05 was considered as statistically significant.