Chemicals and reagents
Luria-Bertani broth (Himedia); Ampicillin, NaCl, Phenylmethylsulfonylfluoride (PMSF), MgCl2, APS, DMSO, Ethanol (Mol Bio grade), Isopropanol (Mol Bio grade) were purchased from MP biomedicals; IPTG and Dithiothreitol (DTT) from Calbiochem; MES, BES, SDS from Sigma; EGTA, Protease inhibitor cocktail, Tris base, 40% Acrylamide, TEMED from Invitrogen. For cell culture studies, Dulbecco modified eagle’s media (DMEM), Fetal bovine Serum (FBS), Horse serum, Phosphate buffer saline (PBS, cell biology grade), Trypsin-EDTA, Penicillin-streptomycin, Pierce™ LDH Cytotoxicity Assay Kit (Thermo, cat no 88953), RIPA buffer were also purchased from Invitrogen. MTT reagent, Okadaic acid and TritonX-100 were purchased from Sigma. The coverslip of 0.17 mm was purchased from Bluestar for immunofluorescence. In immunofluorescence and western blot study we used the following antibodies: Beta-actin (Thermofisher cat no. MA515739) Beta Tubulin (BT7R) (Thermofisher, cat no MA516308) and total Tau antibody K9JA (Dako, cat no A0024), pT181 (Invitrogen, cat no 701530) AT8 (Thermo fisher, cat no MN1020), GSK-3β (Thermo fisher, cat no MA5-15109), Phospho-GSK-3β (Ser9) (Thermo fisher, cat no MA5-14873), anti-ApoE (Sigma, cat no. SAB2701946), 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), Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody with Alexa Fluor 594 (A27016) and DAPI (Invitrogen).
Protein expression and Purification
Full length Tau (hTau40wt) in pT7C were transformed and expressed in BL21* cells while HDAC6 in pET28a-LIC was transformed and expressed in BL21 Codon plus RIL cells. Full-length Tau and repeat domain Tau were purified in two steps using cation exchange chromatography and Size-exclusion chromatography. The cells expressing these proteins after transformation were scaled up and harvested. The cells were lysed by homogenization at 15000 KPSI. The lysate was supplied with 0.5 M NaCl and 5 mM DTT and kept at 90°C for 20 minutes to denature all the structured protein. The resulting sample was centrifuged at 40000 rpm for 45 minutes. The supernatant was kept for overnight dialysis in 20mM MES pH 6.8. The dialyzed sample was centrifuged again at 40000 rpm for 45 minutes and the supernatant was filtered and loaded onto Sepharose fast flow (SPFF) column pre-equilibrated with 20 mM MES pH 6.8, 50 mM NaCl. Elution was carried out using 20 mM MES pH 6.8, 1 M NaCl. The fractions collected from cation exchange chromatography containing Tau protein were pooled, concentrated and subjected to Size-exclusion chromatography using 1X PBS, 2 mM DTT in Superdex 75 Hi-load 16/600 column (Gorantla et al. 2017a; Gorantla et al. 2017b). Purification of HDAC6 ZnF UBP was carried out by Ni-NTA affinity chromatography using 50 mM Tris-Cl pH 8.0 with 20 mM Imidazole for wash and 1000 mM imidazole for elution. The sample was dialyzed overnight in 50 mM Tris-Cl pH 8.0, 100 mM NaCl, 2.5 % glycerol to remove imidazole followed by Size-exclusion chromatography using Superdex 75 Hi-load 16/600 column (Ouyang et al. 2012).
Cell viability by MTT assay
The effective concentration of HDAC6 for the subsequent treatments was determined by studying the concentration dependent toxicity studies by MTT assay. 104 neuro2a cells (ATCC CCL-131) were seeded in a 96 well culture plate in DMEM supplemented with 10% FBS and antibiotic penicillin-streptomycin for 24 hours at 37 °C CO2 incubator. The cells were treated with HDAC6 (0- 500 nM) in serum-starved media for 24 hours. MTT at the concentration of 0.5 mg/mL was added to the cells and incubated for 3 hours. The reduction of MTT by cellular enzymes forms formazon crystals, which were dissolved in DMSO, and the colour developed was quantified by reading at 570 nm in a TECAN Infinite 200 PRO plate reader.
LDH assay
The effect of HDAC6 treatment on cell membrane integrity was studied by LDH (Lactate Dehydrogenase) assay. The disruption of cell membrane integrity leads to the leakage of LDH enzyme, which is quantified by an enzymatic reaction giving a colored end product. For performing LDH assay the cells were incubated and treated as mentioned for MTT assay. After the treatment with HDAC 6 supernatant media was used and the assay was performed according to the manufacturer’s protocol. In brief, 50 µL of cell supernatant was incubated with 50 µL of the reaction mixture provided for 30 minutes at room temperature. 50 µL of stop solution was added to each well and the colour developed was measured at 490 nm and background subtraction at 680 nm was done.
Caspase 3/7 activity assay
In order to study the effect of HDAC6 on inducing apoptotic cell death, the activity of executioner caspase 3 was determined by EnzChek™ Caspase-3 Assay Kit. 10000 cells/well cells were seeded in a 12 well culture plate for 24 hours and further treated with HDAC6 (0-500 nM) for 24 hours in serum-starved media. Caspase activity was performed as per manufacturer’s protocol. The cells were lysed with provided lysis buffer in freeze-thaw cycles. The cell debris was centrifuged out and the supernatant was incubated with fluorescent substrate (DEVD-Rhodamine). The fluorescence was quantified at (Ex/Em) 496 /520 nm at different time intervals in TECAN Infinite 200 PRO plate reader.
Cell culture, Immunofluorescence and quantitative analysis
Neuro2a cells from passage number 10-20 were cultured in advanced DMEM supplemented with penstrep-glutamine, anti-mycotic and 10% FBS. For immunofluorescence studies 5X104 cells were seeded on a glass coverslip (Bluestar) in a 12 well culture plate. Cells were given the desired treatment in serum starved media (0.5% FBS) for 24 hours including groups involving 25 nM Okadaic acid (OA) treatment. After incubation period, cells were washed with PBS and fixed with 4% paraformaldehyde. Further cells were washed with PBS thrice and permiabilized using 0.2% Triton X-100. Cells were blocked with 2% horse serum and incubated with primary antibodies in a moist chamber at 4 ºC overnight. Next day, cells were washed thrice with 1X PBS and incubated with alexa fluor labeled secondary antibodies for 1 hour at 37 ºC. The unbound secondary antibody was washed off with three washes of PBS and counterstained with DAPI. The coverslips were mounted in 80% glycerol and observed under 63X oil immersion lens in Axio Observer 7.0 Apotome 2.0 (Zeiss) microscope. The quantification of immunofluorescence intensity was carried out by Zen 2.3 software and mean fluorescence intensity per unit area were determined for various treatment groups in respective number of fields (n).
Statistical analysis
Two-tailed unpaired student t-test was used to determine the significance for experiments involving comparison of two groups (n.s. – non-significant, * indicates P ≤0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001). One way ANOVA was conducted for the levels of phospho-epitopes (pT181 and AT8) to compare different treatment groups. Tukey’s HSD (Honest significant difference) test was performed to compare the significance within groups (Significant at Tukey’s HSD p<0.05). All the experiments were performed in triplicates and analyzed by Sigmaplot 10.0 (Systat software).