Neuronal culture preparation.
All procedures were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals Norway (FOTS 20135149/20157494/20170001). Wistar Hannover GLAST rat pups (n = 328), embryonic day 18 (E18) to postnatal day 10 (P10), were used for neuronal culture preparation.
Briefly, following anaesthesia and decapitation, the brains were rapidly transferred into preparation solution: ice-cold EBSS solution (Gibco, #24010043) containing 0.5% glucose (Sigma, #G8769) and 10 mM HEPES (Gibco, #15630056). Under a dissection microscope, carefully remove the meninges, cut off the medulla oblongata and separate the cerebellum from the pons and the midbrain. Depending on the culture, Purkinje neuron or structural layer, transfer either only the cerebellum or the cerebellum including pones to a 15 mL tube containing 20 U/mL papain (Worthington, #LK003178) solved in preparation solution and warmed up to 36 ◦C. Place the tube into the incubator for 15 minutes at 36◦C with occasionally swirling to digest the tissue. Remove the papain solution carefully with a fire polished Pasteur pipette and stop the digestion by adding pre-warmed stop media (36◦C): advanced DMEM/F12 solution (Gibco, #12634010) containing 0.5% glucose (Sigma, #G8769) and 10% foetal bovine serum (FBS, Gibco, #10500064). After 5 minutes of deactivation, remove the stop media and add 250 µL growth media containing 10% FBS per cerebellum and pipette the tissue/media suspension with a fire polished Pasteur pipette 100X until cells are separated.
3D Support Cell Layer (3D-SCL).
375000 cells/mL from cerebellum including pones were seeded on pre-coated coverslides from Neuvitro (#GG-12-1.5-PDL, 24 well, 500 µL/well; #GG-18-1.5-PDL, 12 well, 1 mL/well; #GG-25-1.5-laminin, 6 well, 2 mL/well). Culture were maintained in 6-,12- or 24-well plates in growth media consisting of 45% advanced DMEM/F12 solution (Gibco, # 126340010), 45% NBM solution (Miltenyibiotec, #130-093-570), 1.5% B-27 serum-free supplement (Gibco, #17504044), 1.5% NB-21 serum-free supplement (Miltenyibiotec, #130-093-566), 1% NaPyruvate (Invitrogen, #11360088), 1% heat-inactivated FBS (Invitrogen, #10500064), 2% Glutamax (Gibco, #35050038), 5 mg/mL D-glucose and 10 mM HEPES (Invitrogen, #15630056) at 36°C. Half of the culture medium was replaced every 7 days.
Purkinje neuron layer.
E18 and P0 derived Purkinje neuron culture: 500000 cells/mL from cerebellum without pones were seeded on the 3D support cell layer of different in vitro ages. P10 derived Purkinje neuron culture: 750000 cells/mL from the vermis of the cerebellum were seeded on the 3D support layer of different in vitro ages. The growth media was supplemented with insulin (Invitrogen, #12585014; 1:250, stock 4 mg/mL), progesterone (Sigma, #P8783, 1:2000, stock 80 mM), insulin-like growth factor 1 (IGF1; Promokine, #E-60840, 1:40000, stock 1 µg/µL) and Protein kinase C inhibitor K252a (Alomone, # K-150; IC50 25 nM). In long-term cultures that were maintained for more than 28 days in vitro the IGF1 and progesterone concentration were reduced to 10 ng/mL and 20 µM, respectively. K252a was supplemented for 21 days before the washout process started, its optimal concentration was experimental evaluated for each tested culture type. Half of the culture medium was replaced every 3.5 (6 well) and 2 (12/24 well) days, respectively. All experiments testing the Purkinje neuron yield dependent on derived tissue age, in vitro age of the 3D-SCL and K252a concentration were performed randomly, containing 3 to 6 probes per experimental setting and 5 independently repeats for each group and condition.
Lentiviral gene editing.
L7 promoter (full length 1005 bp) were custom cloned by SBI System Bioscience into construct pCDH-L7-MCS-copGFP (#CS970S-1) and viral particle with a yield of 2.24 x 109 ifus/mL were produced. Freshly prepared Purkinje neurons of E18 or P0 cerebellum suspended in growth media containing no serum were incubated for 10 minutes at 37°C with 1.22 x 106 viral particle/mL before seeded onto the supplement structure layer containing cover-slip or live cell imaging µ-dish (#80136, 35 mm, Ibidi). Media was changed after 3 days and transfection efficiency evaluated by live cell imaging microscopy 24h post transfection, daily up to 21 days and weekly up to 169 days in culture, respectively. Additional, lentiviral transfection of Purkinje neurons in culture were performed 1 day after feeding at DIV15 and DIV29 by applying 2.5 x 106 viral particle/mL to evaluate the efficiency and effects of age-dependent genetic manipulations. The neuronal development of the GFP expressing Purkinje neurons was followed by obtaining 10 independent 3x3 tile scan using the Zyla camera configuration (2048x2048) with the CFI Plan Apochromat Lambda dry objective 10x0.45 (pixel size 603 nm) or 20x0.75 (pixel size 301 nm) at the Andor Dragonfly microscope system (Oxford Instruments company). The experiments of DIV0, DIV15 and DIV29 were repeated three times.
Immunohistochemical cell type characterisation.
To evaluate Purkinje neuron yield and the distribution ratio of other cell types of the cerebellum, including their synaptic interactions, the culture was washed with pre-warmed 0.1 M PBS (1xPBS; Gibco, #70013016) and fixed with 1.5-4% paraformaldehyde (PFA, pH 6-7.2; ThermoScientific, #28908) containing 0.5% sucrose for 15 minutes at 36°C. Tris-based or citric acid-based heat induced antigen retrieval (pH 9 and pH 6; 45 min, 85°C) 21 were perform when necessary (see Table 1). Culture were quenched with 1xPBS containing 50 mM NH4Cl (PBSN), permeabilised with 0.2% Triton X-100 (Sigma, #T9284) in PBSN (5 min, 36°C), rinsed with PBSN containing 0.5% cold water fish gelatine (Sigma, #G7041)(PBSNG, 3x15 min), and incubated with primary antibody over-night at 4°C in PBSNG containing 10% Sea Block (SB; ThermoScientific, #37527), 0.05% Triton X-100 and 100 µM glycine (Sigma, #G7126) to visualise the different cerebellar cell types, including Purkinje neurons and their synaptic interactions (Table 1). The cover-slips were rinsed with PBSNG (3x20 min) and incubated with highly cross-absorbed donkey secondary antibodies conjugated to CFTM488/594/647-Dye (1:400; Biotium, #20014, #20115, #20046, #20015, #20152, #20047, #20074, #20075, #20169, #20170) for 2 hours at 22°C in PBSNG containing 2.5% SB. To remove unbound secondary antibody cover-slips were rinsed with PBSN (3x20 min), and briefly tipped into MilliQ water before mounted in hardening ProlongTM Glass Antifade Reagent (Invitrogen, #P36981) onto cover-slides. After 2 days of hardening at 18-21°C in the dark, cover-slides were stored at 4°C until imaging.
Purkinje neuron count and imaging.
Purkinje neurons were counted manually and blind by screening the cover-slips using a Leitz Diaplan Fluorescence microscope equipped with CoolLED pE-300white. For dendritic tree branch analysis and determination of maturity and synaptic interaction, 10 Purkinje neuron Z-stack images per cover-slide were collected in 5 independent and randomized experiments at 0.5-1 µm intervals with the Zyla camera configuration (2048x2048) at the Andor Dragonfly microscope system using either a CFI Plan Apochromat Lambda S LWD 40x1.14 water objective (pixel size 151 nm), 60x1.20 oil objective (pixel size 103 nm) or CFI SR HP Apo TIRF 100x1.49 oil objective (pixel size 60 nm) to detect DAPI and CFTM488/594/647 dye emission and superimposed with Fusion software (Oxford Instruments). 3D surface visualization of synapses was performed using Oxford Instruments analysis software IMARIS 9.3.1 and the filament tracer tool 22.
Dendritic tree branch analysis.
The Purkinje neuron dendritic tree development was evaluated by analysing group dependent 10 Purkinje neurons per experiment in 10 independent experiments towards the order and length of the dendritic arbours by using an open-source ImageJ and Fiji plugin Simple_Neurit_Tracer (Neuroanatomy) 23.
Micro-electrode array (MEA) recordings.
Primary cultures of E18 derived-PNs at a concentration of 500000 cells/mL were plated onto PDL precoated 24 well format plate of the Multiwell-MEA-system (Multi Channel System-MCS, Reutlingen, Germany). Each well contains 12 PEDOT coated gold micro-electrodes (30 µm diameter, 300 µm space, 3 x 4 geometrical layout) on glass base to facilitate visual checking (#890850, 24W300/30G-288). The amplifier (data resolution: 24 bit; bandwidth: 0.1 Hz to 10 kHz, modifiable via software; default 1 Hz to 3.5 kHz; sampling frequency per channel: 50 kHz or lower, software controlled; input voltage range: ± 2500 mV), stimulator (current stimulation: max. ± 1 mA; voltage stimulation: max. ± 10 V; stimulation pattern: pulse or burst stimulation sites freely selectable) and heating element (regulation: ± 0.1°C) is integrated in the Multiwell-MEA-headstage which is driven by the MCS-Interface Board 3.0 Multiboot. The Multiwell recording platform is covered by a mini incubator to provide 5% CO2 and balanced air. Electrophysiological signals were acquired at a sampling rate of 20kHz through the commercial software Multiwell-Screen. Plates were tested every second day for spontaneous activity from day 5 in vitro. Raw voltage traces were recorded for 120 seconds, saved and analysed using offline MCS-Multiwell-Analyzer to calculate spike rate and burst activity, including network properties. Two experimental settings were tested: number 1 recording of spontaneous spike activity in Purkinje neuron culture media (45% advanced DMEM/F12 solution, 45% NBM solution, 1.5% B-27 serum-free supplement, 1.5% NB-21 serum-free supplement, 1% NaPyruvate, 1% heat-inactivated FBS, 2% Glutamax, 5 mg/mL D-glucose, 10 mM HEPES, 16 µg/mL insulin, 25 ng/mL IGF1, 40 µM progesterone, 5 nM K252a) for 63 days and number 2 recording spontaneous spike activity for the first 28 days in Purkinje neuron culture media but then exchanged to organotypic brain slice culture media 15 (30% advanced DMEM/F12 solution, 20% MEM solution (#41090028; Gibco), 25% EBSS solution (#24010043; Gibco), 25% heat-inactivated horse serum (#H1138; Sigma), 2% GLUTAMAX, 5 mg/ml D-glucose and 2% B-27 serum-free supplement) for the remaining 45 days.
Sera were obtained from two untreated patients with gynecological cancer and PCD who had Yo antibodies against CDR2 and CDR2L (anti-Yo1−2) but lacked P/Q-type VGCC antibodies 15. A pool of sera from 100 healthy donors (non-hCDR100p) were used as controls. Sera were not heat-inactivated before use. The sera were stored at the PND Biobank #133/2015 or the Biobank for diagnostic cancer marker #188.05 with approval of the regional ethics committee, Western-Norway.
E x vivo PCD model
Twenty-eight days post-seeding, the culture medium was replaced with medium containing; human serum positive for Yo antibodies (anti-Yo; hCDR2/2L; 4 µL/mL). Purkinje neuron culture was collected 2 or 4 days after commencement of treatment to evaluate the antibody effects (Figure. 2b-c). Each independent experiment included treatment (anti-Yo1−2) and positive (non-hCDR100p) control to account for variations in cell survival between culture preparations. All treatments were performed in triplicate and 5 PNs were analyzed each.