Induced pluripotent stem cell culture and differentiation
The following human induced pluripotent stem cells (hiPSCs) were obtained under Material Transfer Agreement (MTA) from Dr. Stormy J. Chamberlain at the University of Connecticut, Farmington CT: (1) MHC2-10; derived from 3.5 year-old female, control (parental line) line, referred to as UBE3A WT; (2) MHC2-10deltaU, MHC2-10 with a CRISPR engineered homozygous knockout (KO) mutation in UBE3A; referred to as UBE3A KO (3) AG2-3, derived from a 2.5 years-old male Angelman Syndrome patient with large deletion of maternal 15q11-q13; (4) AS2-1, derived from a female Angelman Syndrome patient of unknown age with a large deletion of maternal 15q11-q13. These AS hiPSC lines and the female unaffected control and isogenic UBE3A KO mutant have been described previously (8, 17).
hiPSCs were maintained in Gibco E8 Flex medium on Corning hESC-grade Matrigel diluted 1:1000 in Gibco DMEM/F12. iPSCs were split 1:12 − 1:20 using 50 µM Gibco EDTA diluted in 1X PBS. To begin differentiation to dorsal forebrain fated neural stem progenitor cells (NSPCs), iPSC colonies were lifted for 2 hours at 37°C using collagenase IV diluted to 1 mg/mL in DMEM/F12 and cultured as floating colonies on untreated plates for 24 hours in DMEM/F12 containing 1% N2 supplement and 2% B27 supplement without vitamin A (both Thermo Fisher). After 24 hours, 0.1 µM LDN193189 and 10 µM SB431542 were added to the media. Cells continued to grow as floating colonies for a total of 3 days before being transferred to plates coated with 10 µg/mL Poly-L-Ornithine and 5 µg/mL Laminin for adherent growth. Following ~ 7 days growth under these culture conditions, neural stem progenitor cell (NSPC) rosettes were enzymatically selected by incubating with Gibco Neural Rosette selection media for 1 hour at 37°C, centrifuged for 3 minutes at 300 x g and re-plated on Matrigel in DMEM/F12 medium containing 1% N2 supplement, 2% B27 supplement without vitamin A, and 4 ng/mL recombinant human FGF basic.
NSPCs cultures were expanded and split 1:3 to 1: 5 several times using StemPro Accutase until pure colonies of NSPCs were obtained. To begin differentiation to dorsal forebrain neurons, NSPCs were plated at a density of 0.5 x 105 cells per well of a 24-well plate. Twenty-four hours after plating, NSPCs were transferred to media consisting of STEMCELL Technologies BrainPhys™ media, containing 2 mM GlutaMAX-l supplement, 1.8% bovine serum albumin, 2% hESC supplement, 10 ng/mL BDNF, 10 ng/mL GDNF, 300 µM GABA, and 0.81 mM calcium chloride. On day 4 of neuronal differentiation, this media was supplemented with 10 µM BAY K8644, 10 µM DAPT, and 2 µM PD0332991. On day 7 of neuronal differentiation, cultures were transferred to media containing BrainPhys™ media, containing 2 mM GlutaMAX- l supplement, 1.8% bovine serum albumin, 2% hESC supplement, 10 ng/mL BDNF, 10 ng/mL GDNF, 0.81 mM calcium chloride, and 10 µM BAY K8644, and maintained in this media until endpoint.
For immunocytochemistry and whole-cell patch-clamp recordings, NSPCs were plated at a density of 0.5 x 105 cells per well of a 24 well plate, each containing a glass coverslip coated in hESC-grade Matrigel. Cells were differentiated to neurons for 14–21 days, as described above.
Liquid chromatography tandem mass spectroscopy (LC-MS/MS)
Three independent culture replicates of UBE3A WT and UBE3A KO hiPSC-derived cortical NSPCs were maintained in NSPC medium or differentiated to neurons for 21 days before cell lysis. Cell lysis was performed with 110µL of urea lysis buffer (8M urea, 50mM Tris.HCl pH8, 150mM NaCl) using a QSonica sonic probe with the following settings: Amplitude 50%, Pulse 10 x 1s. 1 on 1 off. The lysate was incubated at room temperature for 1hr with mixing at 1000 rpm in an Eppendorf ThermoMixer and then clarified by centrifugation at 10,000g for 10 minutes at 25°C. The cleared lysate was transferred to 1.5mL LoBind Eppendorf tubes. Protein quantitation was performed using a Qubit protein assay (Invitrogen). 50µg of each lysate was moved and digested with the following protocol: 1) Reduced with 14mM dithiothreitol at 25°C for 30 minutes followed by alkylation with 14mM iodoacetamide at 25°C for 45 minutes in the dark; 2) Digested with 20µg sequencing grade trypsin (Promega) at 37°C overnight. The final digest volume was 1mL adjusted with 25mM ammonium bicarbonate; 3) The digest was cooled to 25°C and terminated with 5µL of formic acid and desalted using solid phase extraction on a Waters Oasis HLB µElution plate. Eluted samples were frozen and lyophilized.
For Tandem Mass Tag (TMT) labeling, samples were reconstituted in 100 µL 70/30 200 mM HEPES, pH 8/acetonitrile (v/v). TMT labels were reconstituted in 40 µL acetonitrile and incubated at room temperature for 15 minutes. A unique label was assigned to each sample, and 15 µL of the preparation was added, by mixing at 300 RPM at room temperature for 90 minutes. Reactions were quenched by adding 5 µL of 5% hydroxylamine and incubated for 15 minutes at room temperature. Equal amounts of labeled peptides were then combined and desalted using solid phase extraction. Samples were then frozen and lyophilized.
250µg of each labeled pooled sample was next separated into 96 discrete fractions using an Agilent 1100 HPLC system equipped with a diode array and fraction collector. The HPLC column was a 2.1 x 150mm WatersXBridge column and peptides were eluted with a basic (pH10) reverse phase buffer system. Fractions were pooled based on the numbered matrix below to yield 12 pooled samples numbered 1–12. Each pooled sample was then lyophilized and reconstituted in 0.1% TFA to produce twelve samples for LCMS/MS analysis.
20% of each pooled sample fraction (the equivalent of ~ 2.5µg of digested sample) was analyzed by nano LC-MS/MS with a Waters M-class LC system interfaced to a ThermoFisher Fusion Lumos. Peptides were loaded on a trapping column and eluted over a 75µm analytical column at 350nL/min; both columns were packed with Luna C18 resin (Phenomenex). Each fraction was analyzed with a 2hr gradient (24 total LCMS/MS time). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 120,000 FWHM and 50,000 FWHM for MS and MS/MS respectively. APD was enabled and the instrument was run with a 3s cycle for MS and MS/MS.
LC-MS/MS data were processed with MaxQuant version 1.6.14.0 (Max Planck Institute for Biochemistry) which incorporates the Andromeda search engine. The Andromeda settings were as follows: (1) Enzyme: Trypsin/P (2) Database: SwissProt Human (3) Fixed modification: Carbamidomethyl (C) (4) Variable modifications: Acetyl (Protein N-term), Oxidation (M) Missed cleavages: 2 (5) Reporter ion tolerance 0.003Da. The MaxQuant output was further processed using Microsoft Excel.
HEK 293T and HeLa cell culture
HEK 293T cells were maintained in DMEM with 10% FBS and 1X Gibco Penicillin-Streptomycin. Cells were split 1:3 − 1:5 by incubating in trypsin-EDTA (0.25%) for 3–5 minutes at room temperature for HEK 293T cells and 37°C for HeLa cells. For immunocytochemistry, 1.25 x 105 cells per well were plated in 24 well plates in 24 well plates on glass cover slips. Cells were plated at a density of 6.25 x 105 cells per well of a 6 well plate or
1.25 x 105 cells per well of a 24 well plate for transfection.
Plasmid Mutagenesis Transfection
The HA epitope N-terminal-tagged, full-length human UBE3A expression construct (HA-E6AP, Addgene plasmid #8657) was used as a template for PCR mutagenesis using the QuickChange Site-Directed Mutagenesis kit (Stratagene) as per the manufacturer's instructions. Forward and reverse primers contained the desired mutation. Reactions were performed in a total volume of 50 µl containing 100 ng ataxin-3 plasmid DNA, 125 ng of each forward and reverse primer, dNTPs, 7% dimethylsulfoxide and 2.5 U Pfu Turbo DNA polymerase. Synthesis of mutated DNA was performed in a temperature cycler (Applied Biosystems, GeneAmp PCR System 2400). Aliquots of 50 µl E. coli XL1 blue supercompetent cells were transformed with 1 µl DpnI-treated DNA and colonies selected on LB ampicillin agar plates. Plasmid DNA was isolated from random colonies and mutagenesis confirmed by DNA sequencing. Primers used were: TTG TTT TGG TTT AAT CCG AGC TCT GAA ACT GAG GGT CAG TTT ACT CTG ATT G and ACT GAC CCT CAG TTT CAG AAG AGC TCG GAT TAA ACC AAA
ACA ATT TTG TAG ATT CAT CGT. The PCR was per formed using PfuTurbo DNA Polymerase (Agilent). according to the manufacturer’s instructions and the PCR cycle: 98°C for 2min followed by 18 cycles of 98°C for 30sec, 52°C for 15sec, 68°C for 8min, followed by 68°C for 7min extension to end. The PCR product contains the nucleotides TCT instead of TTT at codon 583 of human UBE3A isoform resulting in the substitution of a serine for a phenylalanine in human UBE3A isoform 1, a F583S mutation. DDK or 6xHis epitope N-terminal-tagged, full- length human COMT, GRIPAP1, NPTX2, PACSIN1, VAT1 and TH expression plasmids were purchased from GenScript Biotech.
Twenty-four hours after HEK293T or HeLa cells were plated, or once cells reached 50–80% confluency, cell cultures were co-transfected with expression plasmids for HA-UBE3A and DDK-candidate proteins plasmids using the Thermo Fisher Lipofectamine 3000 kit, Gibco Opti- MEM with 1.25-5 µg of plasmid DNA added dropwise to the media.
Protein lysate preparation
Cells were washed with 1X PBS once, then scraped in fresh PBS and collected in a 1.5 mL centrifuge tube. Cells were centrifuged for 5 minutes at 1000 RPM at 25°C. For each well of a 6 well plate, 250 µL of cold lysis buffer was added to the cell pellet, and then the cell mixture was resuspended by vortexing. Cells were incubated in lysis buffer for at least 10 minutes, on ice, then sonicated at 4°C to solubilize the cell suspension. Lysates were then centrifuged for 15 minutes at 10,000 RPM at 4°C. For immunoprecipitation, cells were lysed using an NP-40 IP lysis buffer, which contained 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1% NP-40, and 1X Thermo Fisher protease inhibitor cocktail. All other cells were lysed using Fisher Scientific 1X RIPA buffer (50 mM Tris-hydrochloride, 150 mM sodium chloride, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) with 1X Thermo Fisher protease inhibitor cocktail.
Immunoprecipitation
Immunoprecipitation (IP) assays were completed using the Thermo Fisher DynaBead magnetic bead system. For each IP, 500 ng of lysate was precleared by rocking end-over-end with 10 µL of beads for 30 minutes at room temperature. Beads were then removed. One to five µg of primary antibody was added to each lysate, and lysates were incubated overnight at 4°C, rocking end-over-end. The following day, the lysate was incubated with 30 µL of beads for 3 hours, rocking end-over-end at room temperature. Supernatant was then removed from the beads, and 30 µL of sample buffer with β-mercaptoethanol was added to the beads. Prior to running on a gel for SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis), samples were boiled at 95°C for 5 minutes.
Western blot
Samples were prepared by mixing 20 µg of protein into 3% β-mercaptoethanol in laemmli buffer to 25 µL. They were boiled at 95°C for 5 minutes before being separated by Sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Novex 4–12% Tris-Glycine mini gels using Thermo Fisher Tris-glycine running buffer. Gels were transferred using the BioRad TransBlot Turbo semi-dry transfer system. Blots were blocked in 5% milk in TBST or 5% bovine serum albumin (BSA) in TBST and incubated in primary antibody diluted 1:250-1:1000 overnight at 4°C with rocking. Following 3 x 5 minute washes in TBST at room temperature, blots were incubated in secondary antibody diluted 3:10,000 in 5% milk or BSA for 1 hour at room temperature, with rocking. Secondary antibodies were horseradish peroxidase (HRP)- conjugated goat anti-mouse or goat anti-rabbit IgG depending on the host species used to generate the primary antibody. Blots were developed using Pierce ECL Plus Western blotting substrate for 2 minutes at room temperature. Western blots were imaged using chemiluminescence using an Azure system, and densitometry measurements were quantified using ImageJ software.
In vitro ubiquitination assay
A master mix of UBE1, UBE2L3, ubiquitin, Mg-ATP, 1X E3 ligase buffer (all R&D Systems) were combined and incubated with shaking at 800 rpm at 37°C for 2 hours. Recombinant 6xHis-
UBE3A (R&D Systems) and recombinant 6xHis-tagged candidate UBE3A substrate proteins (GenScript) were then combined with the E1-E2-Ubiquitin master mix for a total of 30 µL per reaction, then incubated with shaking at 37°C for 1 hour. Final concentrations of each component are as follows: 0.17 µM UBA1, 1.28 µM UBE2L3, 75 µM ubiquitin, 10 µM Mg- ATP, 0.8 µM PACSIN1, 0.65 µM GRIPAP1, and 1.5 µM COMT. 10X E3 ligase buffer was prepared by combining 500 mM HEPES, pH 8.0, 500 mM NaCl, and 10 mM TCEP in water. The reaction was then terminated with 2X SDS-PAGE sample buffer. Recombinant His- GRIPAP1, His-PACSIN1 and His-COMT proteins were isolated by Genscript.
Click-IT™ AHA (L-Azidohomoalanine) labeling of proteins
Human embryonic kidney HEK293T cells with CRISPR/Cas9-mediated knockout of endogenous UBE3A (18)(kindly donated by Ype Elgersma, Erasmus MC, Rotterdam, The Netherlands) were maintained at 370C with 5% CO2 in regular growth media (DMEM, Gibco, Cat. No. 11995-065, supplemented with 10% FBS, Gibco, Cat. No.16000-044, and 1X Pen Strep Glutamine, Gibco, Cat. No.10378-016). Cells were plated in 10cm culture dish counting 1.5x106 cells per dish. Cells were transfected with 0.5µg of plasmid DNA per dish using Lipofectamine™ 3000 Transfection Reagent Kit (Invitrogen, Cat.No. L3000008) according to the manufacturer’s protocol. For AHA-treatment, transfected cells were transferred to six-well plates treated with Poly-D-Lysine (Gibco, Cat.No. A3890401) following the manufacture’s protocol.
Transfected UBE3A KO HEK 293T cells were trypsinized (0.05%Trypsin-EDTA, Gibco, Cat. No.25300-054) and transferred subconfluently to six-well culture plates pre-treated with Poly-D- Lysine (Gibco, Cat.No. A3890401) 24 hours after transfection. The next day, cells were gently washed with warm PBS (Gibco, Cat. No. 20012-027) and cultured in methionine-free media (RPMI Medium 1640 L-Methionine free, Gibco, Cat. No. A14517-01) supplemented with 10% dialyzed fetal bovine serum (Gibco, Cat. No. A33820-01) for 1 hour at 370C to deplete the intracellular methionine reserves. Following starvation, cells were cultured in methionine-free media supplemented with 40 µM AHA (Click-iT L- azidohomoalanine, Invitrogen, Cat. No.C10102) for 1 hour at 370C. After an hour of AHA treatment, cells were gently washed twice with warm PBS and cultured in regular growth media. Cell samples were collected at time points: 0h (right after AHA treatment) and 5h.Collected cells were pelleted by centrifugation at 11,000g for 5 min and stored at -800C until use.
Cell pellets were lysed in 1% SDS, 50mM Tris-HCl, pH 8.0 plus a cocktail of endogenous protease inhibitors (Complete Ultra Tablets, Roche, Cat. No. 05892791001) by sonicating with
6–10 pulses (Ultrasonic Processor model CV18, Cole Parmer). The Click-iT Cell Reaction Buffer Kit (Invitrogen, Cat. No.C33372) was used to label AHA-incorporated proteins with biotin according to the manufacturer’s instructions. In brief, samples were treated with Click-iT reagents and incubated at room temperature for 20 minutes using a rotator. In order to isolate AHA-labeled protein from the protein suspension, the samples were incubated with 100 µL of pre-washed NeutrAvidin Agarose Resin (Thermo Scientific, Cat.No. 29200) for 2 hours at room temperature using a rotator. Beads were precipitated by centrifugation at 6000g for 30sec and washed 2–3 times with PBS.
In order to elute bound biotinylated proteins from the beads, the samples were quenched with 2X Laemmili Sample Buffer (Bio Rad, Cat.No. 1610737) and reducing agent (2-Mercaptoethanol, BioRad Cat.No. 1616710) and boiled at 95 0C for 5 min. Proteins were separated by SDS-PAGE (4–20%) and transferred to PVDF membranes (Bio Rad, Cat. No. 1704272), then the blots were incubated in 5% BSA blocking solution (Bovine Serum Albumin, Sigma-Aldrich, Cat. No.
A7906). The membranes were probed with the primary and secondary antibodies (PACSIN1 polyclonal antibody, Invitrogen, Cat. No. PA5-28852; Goat anti-mouse IgG antibody, Sigma- Aldrich, Cat. No. AP127P). Bounded antibodies were detected and imaged by SYNGENE imaging system G-Box (Chemi XRQ model) using chemiluminescence kit (Clarity Western ECL substrate kit, Bio-Rad, Cat. No. 1705061).
Whole-cell patch-clamp recordings
Borosilicate glass capillaries were pulled on a horizontal Flaming/Brown style puller ( Sutter Instruments) and pipettes were filled with internal pipette solution (130 mM potassium gluconate, 15 mM potassium chloride, 5 mM HEPES, 1 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP, pH 7.3). Neurons grown to 14–21 days on glass coverslips, as described above, were placed in a bath of HEPES buffer (135 mM sodium chloride, 30 mM glucose, 10 mM HEPES, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, pH 7.3).
Recordings were obtained with a MultiClamp 700B patch clamp amplifier (Molecular Devices) controlled by pClamp 10 software. After achieving a giga ohm seal using gentle suction, additional brief suction pulses were applied to break the cell membrane under the pipette and establish whole-cell recording conditions. Initial resting membrane potential was measured.
Switching to voltage clamp, the size of the sodium and potassium currents was measured by stepping the membrane potential for 100 ms from a holding potential of -70mV to -50 mV and increasing that step by 5 mV to a final step up to + 45 mV. The maximal amplitude of the fast inward component was taken as sodium current and the average size of the outward current for the last 20 ms of the final step pulse was considered the size of the delayed rectifying potassium current. To test for the presence of spontaneous post-synaptic activity, neurons were recorded gap-free for 3 minutes at a holding potential of -70 mV. The cell was considered synaptically connected when the presence of any, even a single, clear post synaptic current, identified by shape size and time course was observed.
In current clamp mode, holding the membrane potential around − 70 mV, current was injected in 2 pA steps for 1 second each, to determine a maximum number of evoked action potentials. An active voltage response was considered an action potential when the peak crossed 0 mV. A final resting membrane potential was recorded, and initial and final membrane potentials were averaged. Cells were fluorescently labeled by adding 2 µl of a 5 mM stock of membrane impermeant Alexa Fluor 488 hydrazide Sodium salt (Invitrogen) to 1 ml of internal solution for a final concentration of 10 µM. Following the electrophysiological recordings, the cells were imaged on a Zeiss 7 MP microscope with a 40x objective by acquiring an image stack at 1 µm z- intervals to cover all processes in the visual field in 3-D.