Chemicals and Primary antibodies
Luria-Bertani broth (Himedia); Ampicillin, NaCl, Phenylmethylsulfonylfluoride (PMSF), MgCl2, APS, DMSO, Ethanol (Mol Bio grade), Isopropanol (Mol Bio grade) and methanol (Mol Bio grade) were purchased from MP biomedicals; IPTG and Dithiothreitol (DTT) from Calbiochem; MES, BES, SDS, α-Linolenic acid (ALA) (L2376) from Sigma; EGTA, Protease inhibitor cocktail, Tris base, 40% Acrylamide, TEMED from Invitrogen. For cell culture studies, the N9 microglial cell line no. is CVCL- 0452, Roswell Park Memorial Institute (RPMI), Fetal Bovine Serum (FBS), Horse serum, Phosphate buffer saline (PBS, cell biology grade), Trypsin-EDTA, Penicillinstreptomycin, RIPA buffer were also purchased from Invitrogen. MTT reagent and TritonX-100, Trypan -Blue were purchased from Sigma. The coverslip of 12 mm was purchased from Bluestar for immunofluorescence and copper-coated carbon grids for TEM analysis were purchased from Ted Pella, Inc. In immunofluorescence and western blot study we used the following antibodies: Beta-actin (Thermofisher cat no. MA515739), Anti Alpha Tubulin Antibody Clone DM1A (Thermofisher cat no-62204), ARP2MONO (Thermofisher cat no- 703394), Tau Monoclonal antibody (T46) (Thermo cat no-136400), Anti-Iba-1 (Thermo cat no-PA527436), Rab5 (cell signaling, cat no 3547S), Rab7 (Santa Cruz Biotechnology, cat no Sc10767), LAMP-2A (Thermofisher cat no-51-2200), anti-mouse secondary antibody conjugated with Alexa Fluor-488 (Invitrogen, cat no A-11001), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody with Alexa Fluor 555 (A-21428), GOXMS ALEXA FLOUR 488 goat anti rabbit (Thermofisher- cat no A28175) DAPI (Invitrogen), Goat Anti Mouse secondary antibody Peroxidase conjugated (Thermo fisher 32430), Prolong Diamond antifade (Thermofisher cat no- P36961).
Protein expression and purification
Full-length wild type Tau protein (hTau40wt) was expressed in BL21* cells with 100 µg/ml of ampicillin antibiotic selection and purified with two-step chromatography methods, cation-exchange chromatography and size-exclusion chromatography (Gorantla, MiMB, 2018). Cells were grown at 37°C, scaled up and harvested after induction with 0.5 mM IPTG for 4 hours. Cells were subjected to homogenization to produce cell lysate at 15000-psi pressure. The cell lysate was subjected to 90°C heating in presence of 0.5 M NaCl and 5mM DTT for 20 min to denature structured proteins. The supernatant was collected after centrifugation at 40000 rpm for 45 minutes then put through dialysis overnight in 20 mM MES buffer supplemented with 50 mM NaCl. The supernatant was obtained again after centrifugation at 40000 rpm for 45 min passed through cation-exchange chromatography. Sepharose fast-flow column was used for chromatography, using 20 mM MES buffer and 50 mM NaCl (Buffer A). Elution was done with 20 mM MES buffer and 1 M NaCl (Buffer B). A fractions containing Tau proteins were collected after cation exchange chromatography, it was then concentrated and subjected to size-exclusion chromatography. Size-exclusion chromatography was carried out in the Superdex 75 Hi-load 16/600 column in 1X PBS supplemented with 2 mM DTT. A fractions containing Tau were collected, pooled, concentrated and the concentration of protein was determined with BCA (Bicinchoninic acid assay) assay.
Tau protein undergoes aggregation in presence of poly-anionic reagent such as heparin, arachidonic acid, etc., it is observed by the transition of random coiled structure to the β-sheet formation in protein . In this study Tau aggregation was induced by heparin (MW-17500 Da) in the ratio of 1:4 heparin to Tau along with other additives 20 mM BES buffer, 25 mM NaCl, 1 mM DTT, 0.01% NaN3, PIC. The effect of ALA on Tau aggregation was measured by Thioflavin S (ThS) fluorescence assay. ThS is a homogeneous mixture of methylation product of dehydrothiotoluidine in sulfonic acid, which can bind to β-sheet structure. Aggregation kinetics of Tau was studied with 2 µM of Tau and ThS in 1:4 ratios. The excitation wavelength for ThS is 440 nm and the emission wavelength is 521 nm, further analysis of data was done using Sigmaplot 10.0.
Transmission electron microscopy
Morphological analysis of Tau fibrils and ALA vesicles were studied by transmission electron microscopy (TEM). 2 µM Tau sample was incubated on 400 mesh, carbon-coated copper grid and stained with 2% uranyl acetate. For ALA vesicles working concentration of 40µM was taken for grid preparation. The images were taken with TECNAI T20 120 KV.
Conformational changes in Tau from random coiled structure to β-sheet conformation on aggregation of protein was studied using CD spectroscopy, the spectra was collected as previously mentioned in UV region . The measurement was done in Jasco J-815 spectrometer, cuvette path length was 1 mm , measurement was done in range of 250 to 190 nm, and with a data pitch of 1.0 nm, and scanning speed was kept 100 nm/min . For measurement 3 μM sample concentration was taken in phosphate buffer pH 6.8 all the spectra were taken at 25°C.
N9 (microglia) cells were grown in RPMI media in T25 flask or 60mm dish supplemented with 10% heat-inactivated serum, 1% penicillin-streptomycin antibiotic solution and glutamine for maintain the culture. Cells were passaged on 90% confluence using 0.25% trypsin-EDTA solution after washing with PBS. For western blotting experiment cells were seeded in 6 well plate. For α-Linolenic acid preparation, previously published protocol was followed . Briefly, ALA was dissolved in 100% molecular biology grade ethanol and solubilized at 50°C in the stock concentration of 20 mM. The fatty acid solution was prepared freshly before every experiment. According to the previous studies 40 μM was the working concentration of ALA for carrying further experiments. The final concentration of ethanol in cell culture media was maintained below 0.5%.
To study the effect of ALA on microglial phagocytosis, N9 cels were treated with extracellular 1 µM monomer and aggregates along with 40 µM ALA. For the immunofluorescence experiemnt (25,000 cells/well),N9 cells were seeded on 12 mm glass coverslip in 24 well-plate. The cells were then incubated with 1 µM Tau monomer and aggregates along with 40 µM ALA for 24 hours. To compare the internalization ability the controls of 1 μM Tau monomer and aggregates alone for the comparative studies with ALA treatment, 40 μM ALA alone to check morphological changes in N9 cells and cell control (without treatment) were kept. The coverslips were then fixed and stained for immunofluorescence analysis with antibodies Tau T-46 (1:400) and Iba-1 (1:500). The mounting of coverslips were done with mounting media (80% glycerol in 1X PBS). The intracellular intensity of microglia were calculated from fluorescence images to quantify the internalization. The representation of intracellular intensity was done as an intensity/μm area.
To study the degradation of intracellular Tau after phagocytosis we have targeted early and late endosomal markers and lysosme marker for final degradation process. The treatment was done as previously mentioned, after 24 hours of exposure cells were fixed and stained for immunofluorescence analysis. The analysis of the process of degradation was done co-localization of internalized Tau with Rab 5 (1:200), Rab7 (1:200) and LAMP-2A (1:500). The intracellular intensity of Rab 5, 7 and LAMP-2A were studied as intensity/ μm area to understand the expression of proteins on ALA exposure. The colocalization of internalized Tau was studied with 3-D and orthogonal analysis of immunofluorescence images.
Wound scratch assay
To study the migration of microglia wound-scatch assay was performed. For the assay, (5,00,000 cells/well) N9 cells were seeded in a 6-well plate and maintained in RPMI media for 24 hours till the confluency reached to 80%. Scratch was created with sterile 200 μl pipette tip, followed by treatment with groups as mentioned previously. Cells were incubated further for 24 hours to study the migration of N9 cells into the wound. A number of cells migrated into the wound were calculated for 5 different areas of culture and the average was calculated to quantify the migration.
MTOC reorientation analysis
To study immunofluorecence experiment (25,000 cells/well) N9 cells were seeded on 12 mm coverslips in 24- well plate. The desired treatment of Tau monomer, aggregates and ALA was given to cells for 24 hours and fixed for immunofluorescence staining. The MTOC positions were analyzed by β-tubulin (1:200) staining. The anterior, posterior and lateral positions of MTOC were counted with respect to the nucleus denoted by DAPI stain. The percentage of MTOC positions were calculated in 10 different fields.
N9 cells were passaged in RPMI media supplemented with 10% FBS and 1% penicillin-streptomycin. For immunofluorescence studies, 25,000 cells were seeded on 12 mm coverslip (Bluestar) in 24 well plate. Supplemented with 0.5% serum-deprived RPMI media for the desired treatment. The treatment was given for 24 hours. Cells were then fixed with chilled absolute distilled methanol for 20 minutes at -20°C then washed with 1X PBS thrice. Permeabilisation was carried out using 0.2% Triton X-100 for 15 Minutes, washed three times with 1X PBS followed by blocking with 2% serum in 1X PBS for 1 hour at room temperature. Primary antibody treatment was given to cells overnight at 4°C in 2% serum in 1X PBS in a moist chamber. The next day, cells were washed with PBS thrice. Then incubated in the desired secondary antibody in 2% serum at 37°C for 1 hour. Further cells were washed with 1X PBS 3 times and counterstained with DAPI (300 nM). Mounting of coverslip was done in mounting media (80% glycerol). Images were observed under a 63x oil immersion lens in Axio observer 7.0 Apotome 2.0 Zeiss microscope.
Confocal- Super-resolution microscopy analysis
To study the actin structures associated with migration, phagocytosis in presence of ALA Zeiss LSM 980 with Airy scan 2 in super-resolution mode was used. The immunofluorescence staining for the previously described conditions were carried out with β-Actin (1:500) and Iba-1 (1:500) proteins to study the microglia activation and actin structures. The super-resolution mode helped to resolved and understand the minute cell structures such as lamellipodia, filopodia, membrane ruffling and polarization state of microglia. The image processing was carried out with Zeiss ZEN 2.3 software.
For detection of protein levels in cells (3,00,000 cells/well) N9 Cells were seeded in 6 well plate and after the desired treatment for 24 hours. Treatment exposure followed by washing with 1X PBS. Cell lysis was carried out using radioimmunoprecipitation (RIPA) assay buffer containing protease inhibitors for 20 min at 4°C. The cell lysate was centrifuged at 12000 rpm for 20 minutes. Protein concentration was checked by using Bradford’s assay and equal amount of 75 µg total proteins for all the treatment groups were loaded on polyacrylamide gel electrophoresis of range 4-20% and the gel is electrophoretically transferred to polyvinylidene difluoride membrane and kept for primary antibody Rab5, Rab7, LAMP-2A, Iba-1 (1:1000)binding for overnight at 4°C. After the incubation washing of blot was carried out three times with 1X PBST (0.1% Tween-20). The secondary antibody were incubated for 1 hour at RT. Then the membrane was developed using chemiluminescence detection system. The relative quantification of protein was carried out with loading control β- Actin (1:5000) in each treatment group.
All the experiments have performed 3 times. The data is analyzed using SigmaPlot 10.0 and the statistical significance was calculated by student’s t-test (ns- non-significant, * indicates P≤0.05, ** indicates P≤ 0.01, *** indicates P≤0.001). The quantification of levels of intracellular proteins in immunofluorescence experiments was carried out by measuring the absolute intensity of protein and the corresponding area of microglia with Zeiss ZEN 2.3 software for image processing.