3.8.1. Reagents and Chemicals.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), and all cell culture materials were purchased from ThermoFisher Scientific (Waltham, MA) unless otherwise noted.
All primers were purchased from Integrated DNA Technologies (Coralville, IA), and all molecular biology supplies were purchased from New England Biolabs (NEB, Ipswich, MA) unless otherwise indicated.
Fig. 3.5. Proposed Mechanism for the Role of Decreased Cdk5 O-GlcNAcylation in AD Pathogenesis.
Decreased O-GlcNAc on Cdk5 S46 may lead to an increased association of Cdk5 with activator proteins p25/35 and/or p29/39, resulting in higher activity. This, in turn, results in the increased phosphorylation of pathogenic proteins such as tau and APP which eventually leads to the
formation of NFTs and Aβ plaques, respectively. Increases in phoshpo-tau and APP may have even more drastic effects in the context of decreased O-GlcNAcylation seen in AD, as O-GlcNAcylation is known to both oppose phosphorylation and stabilize proteins against aggregation. Adapted with permission from Yuzwa et al. 2014.57
Cdk5
Cdk5
3.8.2. Neuron and Cell Culture.
Primary mouse cortical neurons were harvested as previously described,36 plated on poly- d-lysine coated plates, and cultured in Neurobasal medium with 1% penicillin-streptomycin (P/S), 2mM GlutaMAX Supplement, and GS-21 (MTI-GlobalStem, Gaithersburg, MD). Every 2-4 days, half of the media was changed for fresh culture media as outlined above. Neurons were allowed to grow for 7 days before transduction with lentivirus. Otherwise, they were cultured for the lengths indicated in the text. For KCl stimulation experiments, neuronal activity was first silenced by 24 hours treatment with 10 μM tetrodotoxin (Tocris Biosciences, Bristol UK) and 100 μM D-AP5 (Tocris Biosciences). Neurons were then treated with KCl (60 mM) or vehicle for 2 hours and subsequently lysed with either RIPA buffer (for IP experiments) or 2% SDS in HEPES pH 7.9 (for chemoenzymatic labeling experiments), containing Roche cOmplete protease inhibitor cocktail, 0.1 mM TMG, and 0.25 U/μL benzonase nuclease (Santa Cruz Biotechnology, Dallas, TX). In all cases, the protein concentration was measured using BCA assay (ThermoFisher).
N2A and HEK 293T cells were maintained in Dulbecco's Modified Eagle Medium with 100 U/mL P/S, and 10% FBS. Cells were split 1:8 every 2-3 days as necessary. N2A cell lysis was performed as described above for neurons.
3.8.3. Co-Immunoprecipitation.
Neuronal or N2A lysate, prepared as described above, was diluted to 0.5 mg/ml with Tris- buffered saline (TBS) buffer,75 0.1 mM TMG, and Roche cOmplete protease inhibitor cocktail. 30 μg of protein was saved as input. The remaining lysate was incubated with anti-FLAG magnetic beads (Sigma-Aldrich) or pre-incubated (1 hour) Cdk5 antibody (sc-173, Santa Cruz Biotechnology, 1:10 dilution) and Protein A/G magnetic beads (40 μL, ThermoFisher) at 4 °C
overnight or for 1 hour at room temperature with end-over-end rotation. The following day, beads were washed 3 times with 0.1% Triton-X-100 in TBS and eluted by boiling in 2% SDS in Tris, pH 8. For samples undergoing subsequent WGA affinity chromatography, elution was performed with 150 μg/mL 3xFLAG Peptide (Sigma-Aldrich) in TBS. Inputs and eluents were run on SDS-PAGE and subjected to WB as described below.
3.8.4. Wheat Germ Agglutinin Affinity Chromatography.
Cdk5 eluted from the FLAG resin was immediately incubated with WGA agarose (Vector Laboratories, Burlingame, CA) overnight with end-over-end rotation at 4 °C. The following day, the beads were washed 5 times with 0.1% Triton-X 100 in TBS and eluted with 0.5 M GlcNAc in 0.1% Triton-X 100 in TBS. The elution was then concentrated by vacuum centrifugation and subjected to SDS-PAGE.
3.8.5. Western Blotting.
Primary antibodies were all used at 1:1000 dilution, and secondary antibodies at 1:10000 unless otherwise specified. Samples were diluted with 4x SDS-PAGE buffer (200 mM Tris-HCl pH 6.8, 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol), resolved on a NuPAGE 4-12%
Bis-Tris protein gel (ThermoFisher Scientific), and transferred to Immobilion-FL PVDF membrane (MilliporeSigma, Burlington, MA). Blots were blocked for 1 h at RT with LiCor Odyssey Blocking Buffer (927-50003, LI-COR Biosciences, Lincoln NE) before probing overnight at 4 °C with the following antibodies diluted in blocking buffer: Cdk5 (DC-17, Santa Cruz Biotechnology), FLAG (1:5000, F3165, Sigma-Aldrich), p35 (2860S, Cell Signaling Technologies, Danvers, MA), OGT (DM-17, Sigma-Aldrich), and anti-α-tubulin (Sigma Aldrich,
T9026, 1:3000). Blots were rinsed three times with TBST and then probed with the appropriate secondary antibodies in blocking buffer: anti-mouse AlexaFluor 680 conjugate (A21057, ThermoFisher), anti-rabbit AlexaFluor 680 (A21109, ThermoFisher), anti-rabbit AlexaFluor 790 (A11369, ThermoFisher), or anti-mouse IgG DyLight 800 (A11357, ThermoFisher). Blots were washed 3 x 5 min with TBST and then imaged using an Odyssey Infrared Imaging System (LI- COR Biosciences). Images were processed using ImageStudio v5.2 (LI-COR Biosciences). To calculate O-GlcNAcylation stoichiometry, the ratios of eluents to inputs, weighted by proportion of the total protein in each lane, were used as previously described.76
3.8.6. Cdk5 Activity Assay.
Cdk5 activity was measured using ADP-Glo (Promega). Briefly, Cdk5 was IPed as described above, however, before elution from the FLAG beads, Cdk5 activity towards a well- characterized peptide substrate (sc-3066, Santa Cruz Biotechnology) was measured. Each kinase activity assay was performed in 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.1 mg BSA with the addition of 0.75 μL ultrapure ATP (ADP-Glo) and 0.1 mM Cdk5 substrate peptide (75 μL final volume). As a control, the Cdk5 substrate peptide was omitted or roscovitine (Cell Signaling Technologies), a potent Cdk5 inhibitor, was added. The kinase reaction was left to proceed for 45 min at room temperature with end-over-end rotation. The supernatant was then removed and the amount of ADP assayed with ADP-Glo according to the manufacture’s protocol. Final luminescence values were normalized to the level of Cdk5 in each kinase reaction (after elution from the FLAG beads) as detected by WB.
3.8.7. Chemoenzymatic Labeling.
O-GlcNAcylated proteins from neuronal lysates were labeled as previously described.76 Briefly, lysate containing 150 μg of protein was diluted to 200 μL in water and proteins were precipitated by the addition of 3 volumes of methanol, 1 volume of chloroform, and 2.25 volumes of water followed by centrifugation at 21,130 x g for 5 minutes to reveal a disc-like protein pellet at the aqueous-organic interface. The aqueous layer was removed and the pellet washed three times with 2.25 volumes of methanol. Protein pellets were then resolubilized with 40 µL dissolution buffer (20 mM HEPES, 1% SDS, pH 7.9). Water (49 µL), 5.5 mM MnCl (11 μL), and 80 µL 2.5x GalT labeling buffer (50 mM HEPES, 125 mM NaCl, 5% IGEPAL CA-630, pH 7.9) were added and the solution was vortexed gently before adding 10 µL Y289L GalT (1 mg/mL), and 10 µL of UDP-GalNAz (0.5 mM in 10 mM HEPES, pH 7.9). The reaction was then left to rotate end-over- end at 4 °C overnight. Control experiments were carried out in parallel in the absence of UDP- GalNAz. The following day, proteins were precipitated as before and resolubilized in dissolution buffer (1% SDS in TBS pH 7.6). The following were added (in order) to the redissolved protein:
100 µL H2O, 2.5 µL BTTAA (10 mM, Click Chemistry Tools), 5 µL CuSO4 (50 mM), and 5 µL of biotin-PEG4-alkyne (5 mM, Click Chemistry Tools). The solutions were mixed by gentle vortexing and 4 µL of TCEP was added. The reaction was allowed to react for 1 hour with end- over-end rotation in the dark before being precipitated as before. Proteins were resolubilized in dissolution buffer as described, and 10% of the reaction volume was taken as input. The remaining volume was then incubated with streptavidin magnetic beads (ThermoFisher) for 1.5 hours in the dark. Beads were then washed 5 times with 0.5 ml of low salt buffer (100 mM Na2HPO4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) and 5 times with 1 ml of high salt buffer (100 mM Na2HPO4, 500 mM NaCl, 0.2% Triton X-100). Biotinylated proteins were eluted
by boiling the resin in 50 mM Tris-HCl pH 6.8, 2.5% SDS, 100 mM DTT, 10% glycerol, and 2 mM biotin for 15 min with occasional vortexing.
3.8.8. In-Gel Digestion of Cdk5 for O-GlcNAc Site Mapping.
After SDS-PAGE, the gel was stained and visualized using Imperial protein staining reagent (ThermoFisher Scientific) and destained in doubly deionized H2O (ddH2O). The band corresponding to FLAG-tagged Cdk5 was then excised and stored at -80 °C until in-gel digestion as previously described.77 Briefly, the gel piece was cut into 1 mm squares, reduced with 10 mM DTT for 1 h at 37 ºC, and alkylated with 50 mM iodoacetamide for 45 min at room temperature protected from light. The slices were dried and incubated with 20 ng/uL Pierce Trypsin, or Pierce Chymotrypsin, protease (ThermoFisher Scientific) solution in 100 mM NH4HCO3 (pH 8.0) overnight at 37 ºC. The peptides were recovered using sequential washes with 100 mM NH4HCO3, 1:1 v/v 100 mM NH4HCO3/CH3CN, and 5% formic acid. The recovered peptides were dried and desalted with a C18 ZipTip pipette tip (MilliporeSigma). The desalted peptides were resuspended in 25 μL of 0.2% formic acid for LC-MS/MS analysis.
3.8.9. LC-MS/MS Analysis.
Cdk5 samples were analyzed on a nanoflow LC system, EASY-nLC 1000 (ThermoFisher Scientific), coupled to either a Q-Exactive or LTQ Velos (ThermoFisher Scientific) equipped with a Nanospray Flex ion source.
3.8.9.1. LTQ Velos Analysis (CID/ETD).
Cdk5 peptides (2 μL and 5 μL) were loaded onto a 360x100 μm precolumn (2cm Monitor C18, Waters Milford, MA) prior to separation on a 360x75μm column/tip (9cm BEH 1.7 μm) at 1μL/min at 600 bar constant pressure for 15 μL. The column was heated to 60 °C. The peptides were separated with a 145-min gradient at a flow rate of 250 nL/min. Solvent A consisted of 99.9% water and 0.1% formic acid, and Solvent B consisted of 80% acetonitrile, 19.9% water, and 0.1% formic acid. The gradient was as follows: 0-30% Solvent B (120 min), 30-90% B (2 min), and 90% B (13 min). Full spectra were acquired over m/z 350-1500 in the Orbitrap (60 K resolution at 200 m/z); automatic gain control (AGC) was set to accumulate 50,000 ions, with a maximum injection time of 50 ms. Data-dependent MS2 analysis was performed using a top 5 approach with alternating CID and ETD fragmentation.
3.8.9.2. Q-Exactive Analysis (HCD).
Cdk5 peptides (2 μL) were loaded onto a 360x100 μm precolumn prior (2cm Monitor C18, Waters) to separation on a 360x75μm column/tip (9cm BEH 1.7 μm, Waters) at 1μL/min at 600 bar constant pressure for 15 μL. The column was heated to 60 °C. The peptides were
separated with a 77-min gradient at a flow rate of 250 nL/min. Solvent A consisted of 99.9%
water and 0.1% formic acid, and Solvent B consisted of 80% acetonitrile, 19.9% water, and 0.1%
formic acid. The gradient was as follows: 2% Solvent B (10 min), 2-35% B (60 min), 35-95% B (2 min), and 95% B (5 min). Full spectra were acquired over m/z 350-1500 in the Orbitrap (70 K resolution at 200 m/z); automatic gain control (AGC) was set to accumulate 1,000,000 ions, with a maximum injection time of 50 ms. Data-dependent MS2 analysis was performed as described
previously78 using a top 12 approach with HCD fragmentation at 35 K resolution, a max injection time of 60 ms, and an intensity threshold of 100,000 ions.
3.8.10. LC-MS/MS Data Analysis.
The data was converted to MGF files for Mascot searching using Proteome Discoverer.
ETD and CID spectra were extracted to separate MGF files for searching. The data was then searched against a custom database with the expected sequence of interest and a mouse slice of the UniProt database downloaded in April 2015 with fixed modifications of carbamidomethyl (C) and variable mods of O-GlcNAc (ST), and oxidation (M). Enzyme specificity was Trypsin with 2 missed cleavages and semichymotrypsin with 4 missed cleavages. Mass tolerances were 25 ppm and 0.8 Da for precursor and fragments ions, respectively. CID data and ETD data were searched with instrument parameters of ESI-trap or ETD-Trap, respectively. For HCD, mass tolerances were 25 ppm and 50 mmu for precursor and fragment ions, respectively. Data was then loaded into Scaffold (Proteome Software, Portland OR) and filtered for a 1% false discovery rate at the peptide level.
3.8.11. Lentiviral Plasmid Construction.
Cdk5 shRNA lentiviral plasmid (pLKO.1) was purchased from Sigma-Aldrich (TRCN0000278085). Cdk5-Myc-FLAG in pCMV6 (MR204021) was purchased from Origene (Rockville, MD). Mutations conferring shRNA resistance (1325- CCCTGAGATTGTGAAGTCATTC-1346 to CCCTGAaATTGTGAAGagtTTt) and all serine to alanine mutations were produced using the Q5 Site-Directed Mutagenesis Kit (NEB) according to the manufacturer’s protocols. All primers were designed with the NEBaseChanger software.
shRNA resistant mutant or WT Cdk5 was then linearized by using the Q5 Hot Start High-Fidelity 2x master mix and the pEF-ENTRA A73 entry vector was linearized by digestion with BamHI-HF and XbaI. Linear DNA was then gel purified using the Zymoclean gel DNA recovery kit (Zymo Research, Irvine, CA) and assembled with the NEBuilder HiFi DNA assembly kit according to the manufacturer’s protocol. The Cdk5 entry vectors were then recombined with the pLENTI CMV GFP73 destination vector using the Gateway LR Clonase II Enzyme mix (ThermoFisher) as described by the manufacturer. The final lentivectors were then amplified in Stable Competent E.
coli (New England Biolabs), purified using ZymoPURE II Plasmid Kit (Zymo Research, Irvine, CA), sequenced (Laragen, Culver City, CA), and resolved by 1% agarose gel electrophoresis to ensure plasmid fidelity.
3.8.12. Lentivirus Production.
HEK 293T cells were transfected with the lentiviral plasmids produced above, as well as lentiviral packaging plasmids (ViraPower Lentiviral Packaging Mix, ThermoFisher), using Lipofectamine 3000 (ThermoFisher) at 90% confluency according to manufacturer’s instructions.
The virus was harvested by collecting media every 24 hours for 3 days. Lentiviral particles were concentrated approximately 50 fold at 4 °C using 15-mL 100 kDa MWCO Amicon concentrator tubes (MilliporeSigma), flash frozen immediately, and stored at -80 °C until use. Lentiviral titer was estimated by traducing neurons with a dilution series of lentivirus and estimating transfection efficiency by GFP fluorescence confocal microscopy or determining Cdk5 expression levels compared to endogenous by WB. In all cases, neurons were assayed at least 7 days after transduction.