Hippocampal neuron culture
Hippocampal neural cultures were isolated from the embryonic Sprague–Dawley rats on Day 18–19 following a previously described method [38]. In brief, tissues were dissected and collected in dissection medium, which was made up of balanced salt solution containing Penicillin-Streptomycin (PS, Gibco #15140-122) and HEPES (Gibco #15630-056). The neurons were then dissociated with 0.05% trypsin-ethylenediaminetetraacetic acid (Gibco #25300-054) containing PS and HEPES for 15–20 min. After washing, 3.25 × 105 cells (containing neurons and glial cells) were grown in the 60 mm Petri dishes on the coverslips pre-coated with poly-L-lysine (PLL, P2636-1G Sigma) with neurobasal medium (Gibco #12348-017) containing SM1 supplement (Stem Cell #05711), horse serum (GIBCO, #26050088), and L-glutamine (GIBCO, #25030-024). The culture medium was replaced by serum-free neurobasal medium after five days of culture in vitro. Cells were maintained at 37 °C in 5% CO2 for a maximum of 15 days in vitro.
Animals
Heterozygote A53T-α-syn transgenic mice (B6; C3-Tg (Prnp-SNCA*A53T) 83Vle/J) were originally obtained in breeding pairs from the Jackson Laboratory (004479). Wild-type mice were C57BL/6J purchased from Liaoning Changsheng biotechnology.co., Ltd (China). All mice in the colony were kept under specific pathogen-free (SPF) conditions in a 14 h light /10 h dark cycle, and had free access to food and water. At 10 months, mice were scarified for protein detections.
Transfection and electroporation
Neurons were transfected by the method of calcium-phosphate coprecipitation at DIV9-11 as previously described [38]. Briefly, using a mixture solution of Tris-EDTA (TE), CaCl2, and 2 × HEPES-buffered saline, prepared the precipitates with 0.75–1.25 µg of plasimidic DNA (0.25 µg of Homer1C-Dsred, 0.5 µg wild-type (WT)-/A53T-α-syn, 0.5 µg of GluN2Α/2B-sep-NMDAR/ GluA1-sep-AMPAR for QD tracking and immunocytochemistry, or 0.5 µg of GCaMP6, 0.5 µg of WT-/A53T-α-syn for calcium imaging). Coverslips containing neurons were transferred to 12-well plates and incubated with precipitates in 250 µl of conditional medium in the presence of 2 mM kynurenic acid (Sigma-Aldrich #K3375) for 1–1.5 h. The coverslips were then washed with 2 mM kynurenic acid in unsupplemented neurobasal medium and transferred back to original Petri dishes for four days of overexpression before use.
Neuronal electroporation was performed following the commercial instructions, using the P3 Primary Cell 4D-NucleofectorTM X Kit (LONZA, #V4XP-3024) and 0.75 µg of plasmid DNA (0.25 µg of Homer1C-Dsred and 0.5 µg of WT-/A53T-α-syn) to prepare the electro-buffer. The neurons (5 × 105) were resuspended in electro-buffer and transferred to a Single Nucleocuvette™. Using the Amaxa Nucleofector (Lonza), the cells were electroporated with program DC-104. Afterward, the cells were plated on the coverslips and cultured at the 37 °C in 5% CO2 for 14 days of overexpression before use.
α-Synuclein treatments
The overexpression of α-syn (WT/A53T) was processed by either calcium phosphate transfections or the electroporations. Cells at DIV 9–11 were transfected with 0.5 µg WT-/A53T-α-syn plasmids via the calcium phosphate method for four days of expression. Or dissociated neurons were electroporated with 0.5 µg of WT-/A53T-α-syn plasmids and expressed for 14 days after seeding. To calculate the relative overexpression, we utilized antibody against α-syn to label the α-syn expression in transfected neurons, and the non-transfected neurons in the same coverslip were as basal condition. Then we quantified fluorescence intensity of the α-syn positive signal in every whole neuron, including both cell bodies and dendrites. To perform quantifications, 10 cells were chosen from each condition.
Proteins of α-syn monomer (#RP-003) and PFF (#2015 − 1969) were purchased from Proteos. α-Syn PFF proteins were sonicated at 25% amplitude 1 s and 1 s intervals, 3 min before the application. For an acute exposure, neurons at DIV 13–14 were treated with monomer (1 µM) or α-syn PFF (1 µM) containing conditional mediums for 20 min (QD experiments) or 1.5 h (immunostaining) and washed out before experiments. For chronic exposure, α-syn monomers (1 µM) or PFF (1 µM) were applied to culture medium after transfection at DIV 9–10, and incubated for four days. The concentrations of monomer and PFF were equivalent.
Peptide application
TAT-peptides[39, 40] [TAT-NS: control, scramble sequence; TAT-GluN2B15: TAT-sequence (YGRKKRRQRRR) - GluN2B C-terminus sequence (NGH VYEKLSSIESDV), CASOL] were administered at 5 µM into culture medium of the electroporated neurons, once every other days from DIV7–12, for four applications in total.
Single quantum dots tracking and analysis
Hippocampal neurons at DIV 13–14 were firstly incubated 10 min with polyclonal antibodies against green fluorescence protein (GFP, Invitrogen, Thermo Fisher 1:100 000) to detect overexpressed GluN2Α/2B-sep-NMDAR, followed by 10 min of incubation with QD 655 Goat F(ab’)2 anti-rabbit polyclonal antibodies (Invitrogen, Thermo Fisher Scientific Inc.1:50 000). All the cells were incubated in the neurobasal medium supplemented with 1% bovine serum albumin (BSA) at 37 °C. Coverslips containing neurons were then mounted with Tyrode solution (30 mM D-glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 25 mM HEPES, pH 7.3–7.4) as described previously [38] and placed on a heated chamber for observations. Using a mercury lamp and appropriate excitation/emission filters, QD signals were detected with an electron multiplying charged-coupled device (EMCCD) camera (Evolve, Photometrics). Images were obtained every 50 ms (20 Hz) with up to 500 consecutive frames and processed using the Metamorph software (Universal Imaging Corporation, PA, USA). In each recording session, 3–4 structured dendritic areas were obtained from every coverslip obtained, and the procedure lasted for up to 20–25 min. α-Syn proteins (monomer or PFF) were incubated for 20 min before QD labeling and acquisitions. The calculations of single QD tracking followed the formulas and the rules described previously [38, 39]. Briefly, the instantaneous diffusion coefficient ‘D’ was calculated for each trajectory, from linear fits of the first four points of the mean-square-displacement versus time function using MSD(t) = < r2 > (t) = 4Dt. Using a Vogel algorithm, the two-dimensional trajectories were constructed by correlation analysis between consecutive images. This technique offers high resolution and is capable of measuring the dynamic distribution of GluN2B/2Α-NMDAR at synaptic and extra-synaptic sites. A synaptic area was defined as the area with the transfected synaptic marker Homer1C-Dsred. The diffusion coefficient was calculated from GluN2-QD trajectories were present on both the inside and outside of the synaptic area.
Immunocytochemistry
The following antibodies were used: anti-GFP (Thermo Fisher SCIENTIFIC #A6455), anti-synaptophysin (Synaptic Systems #101 011), anti-α-syn (Santa cruz #sc-12767), anti-myc (Origène #TA150121).
All the secondary antibodies were used at 0.1 mg/mL concentration: anti-rabbit Alexa 488 (Thermo Scientific #A11008), anti-mouse Alexa 647(Sigma-Aldrich #F1804).
To detect the endogenous and overexpressed proteins levels, neurons at DIV14 were fixed with 4% paraformaldehyde (Sigma-Aldrich, #P6148) in phosphate-buffered saline (PBS) containing 4% sucrose (Sigma-Aldrich, #0389) for 15 min at room temperature (RT), followed by the 5 min of permeabilization with 0.25% Triton X-100 in PBS. Non-specific signals were blocked by incubation with 10% BSA (SIGMA) in PBS for 1 h, after which the cell were incubated with primary antibodies for 2–3 h at RT, followed by the secondary antibodies for 1 h at RT. To label the contents of surface glutamatergic receptors (GluN2Α/2B-sep- and GluA1-sep-subunit), dissociated hippocampal neurons were live immunostained for 20 min, using anti-GFP antibody at 37 °C. After the fixation, the cells were incubated with secondary antibodies for 1 h at RT. All the antibodies were prepared in 1% BSA in PBS. All samples were washed at least three times with PBS between the incubations, finally mounted in the Mowiol mounting medium.
Fluorescence microscopic imaging
All the neuronal images were acquired using a Photometrics Quantem 512 camera (EMCCD) and MetaMorph imaging software (Molecular Devices) on an inverted confocal spinning-disk microscope (Leica DMI6000B, Leica), using a Leica HCX PL APO CS 60×/1.4 or 63×/1.4 oil objective. In each individual experiment, images were captured in the same settings (the same laser intensity and exposure time) across all the samples. Images were exported in the grey scale from individual channels and pseudocolor overlays were prepared using ImageJ software. To perform quantifications, 9–11 cells were chosen from each condition from every independent experiment were chosen. For each neuron, two or three dendritic regions were selected for analysis.
Calcium imaging
The experiments were performed similarly as formerly described [41]. Briefly, treated hippocampal neurons were transfected with GCaMP6 at DIV 9–10 for four days and transferred to Tyrode solution at least 3 h before acquisition at DIV 13–14. Before imaging, the cells were incubated in Mg2+-free Tyrode solution supplemented with 5 µM Nifedipine (Tocris) and 5 µM Bicuculline (Tocris) for 15 min. Time-lapse images were obtained every 50 ms for up to 3000 frames. Thereafter, 50 µM D-AP5 was applied directly to the chamber to abolish the calcium transient events. A movie containing another 3000 time-lapse frames was acquired as the baseline for each neuron.
Proteins extraction
10 months old mice (WT and A53T-α-syn transgenic) were harvested and hippocampus were dissected out and stored at − 80 °C. To extract the protein, tissues were thawed and homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, #P0013C) containing 2 mM PMSF (Beyotime, #ST506) and 1% protease inhibitor cocktail (Sigma-Aldrich, #P8340). Following homogenization, samples were centrifuged at 13,000 rpm for 30 min at 4℃. The supernatants were collected as protein samples and stored at − 80℃.
Synaptosomes components were purified from homogenized hippocampus of 10-month-old WT-and A53T-α-syn transgenic mice, following the methods as previously described [39]. Hippocampal tissues (around 20 mg) were thawed in 300 µL fresh TPS buffer (0.32M sucrose 4 mM HEPES buffer, pH7.4) supplemented with a protease inhibitor cocktail with 15 strokes in glass-Teflon homogenizer (500 µL), on the ice. After centrifugation at 1000 g for 8 min at 4℃, the pellet was discarded and the supernatant was centrifuged again at 13,500 g for 15 min at 4 °C. The resulting pellet was washed by TPS buffer once and resuspended in RIPA lysis buffer supplemented with protease inhibitor cocktail and PMSF for 15 min lysis. Finally, samples were collected and kept at − 80℃ for storage.
Western blotting
The protein content was quantified through BCA assay (Beyotime, #P0012) and then added 5 x SDS loading buffer (Beyotime, #P0016) and PBS to gain equal concentration. Before loading, samples were boiled 5 min at 95℃.The extractions containing 15 µg of protein were separated by SDS/PAGE (Tris-Gly, 4%-15% precast gel, Sangon Biotech, #C651104) for 60 min 150 V, transferred onto polyvinylidene difluoride membranes (Millipore, #ISEQ00010) and blocked in 5% skimmed milk (BD, #232100) for 1 h and then immunoblotted with specific antibodies. The following primary antibodies were used: anti-GluN2A Ab (1:1000, Sigma-Aldrich, #04-901), anti-GluN2B Ab (1:5000 Abcam, #ab65783), anti- GluN1 Ab (1:5000, Synaptic Systems, #114011), anti- GluA1 Ab (1:5000, Synaptic Systems, #182003), anti-PSD95 Ab (1:2000 Abcam, #ab18258), anti-synaptophysin Ab (1:5000, Synaptic Systems, #101 011), anti-β-actin Ab (1:10000 Sigma-Aldrich, #A1978), and diluted in TBST 0.5% milk overnight at 4 °C. Corresponding secondary antibodies were used at 1:5,000 in TBST 0.5% milk.
Ethics Statement
Animal experimentation: All experiments were carried out in accordance with the guidelines and regulations of University of Bordeaux and Northeastern University. The animal procedures were approved by the ethical committee of the University of Bordeaux (Laurent Groc experimentation authorization number 3306009) and Northeastern University, China.
Data and statistical analysis
All data and statistical analysis was performed by GraphPad Prism 8.00 (GraphPad Software, Inc., La Jolla, CA). Most non-normal data, such as single-particle tracking and immunocytochemistry of the data were assessed by Mann-Whitney test to compare differences between two groups and by Kruskal-wallis test followed by Dunn’s Multiple Comparison test between three groups. Normal data such as expression levels using unpaired student’s t test. Significance was assessed at p < 0.05 using two-tails tests unless otherwise specified in the figure legends. Symbols used are: *p < 0.05; **p < 0.001; ***p < 0.0001 throughout the manuscript.