Patients, NR1 antibodies, titers, and controls
Cerebrospinal fluid and serum were obtained from randomly selected patients with anti-NMDAR encephalitis (Supplemental Table 1
) among a series of 320 cases. All patients had well characterized clinical manifestations of anti-NMDAR encephalitis, including at least 4 of the following features: prominent psychiatric symptoms, decreased level of consciousness, seizures, dyskinesias, autonomic instability, or hypoventilation. Antibodies to extracellular epitopes of the NR1 subunit of the NMDAR were demonstrated using three different assays, as reported(Dalmau et al., 2008
): immunohistochemistry with rat and human brain, immunostaining of live, non-permeabilized cultures of rat hippocampal neurons, and immunolabeling of HEK293 cells transfected with NR1 or NR1 and NR2 (forming NR1/2 heteromers). We previously reported that patients' NMDA receptor antibodies are IgG1 and IgG3, but not IgM(Tuzun et al., 2009
); therefore we will refer to purified antibodies from patients' serum as purified IgG. CSF from patients with high antibody titer were diluted so that the final titer used in experiments was within the range of undiluted CSF of many patients with this disorder(Dalmau et al., 2008
Control serum or CSF samples were obtained from normal individuals and patients undergoing CSF analysis for a variety of disorders not associated with antibodies to the NMDAR; samples were randomly selected from 1,500 cases negative for NR1 antibodies applying similar test and criteria as above.
Preparation of patient and control CSF and IgG
Patient or control cerebrospinal fluid (CSF) and serum were collected, filtered, and kept frozen until use. CSF from individual patients with high NMDAR antibody titer was diluted 1:15–60 to treat neurons in vitro
, and used undiluted for in vivo
experiments. In some experiments, patient IgG antibodies were purified from serum with protein A/G sepharose columns and used to treat neurons. To prepare patient and control IgG, 2 ml of serum were incubated with a 1 ml bio-spin chromatography column (Bio-Rad) of protein A/G sepharose beads (50:50) for 30 min. on an orbital shaker at 4 °C. After 3 washes with phosphate buffered saline (PBS), eluted with 100 mM glycine, pH = 2.5 and neutralized with Tris-HCl, pH = 8.0, dialyzed against PBS, concentrated in stock solutions of 20 mg/ml, and stored at −80 °C. IgG concentration (~1mg / ml) and pH (7.4) was adjusted prior to use. Each IgG preparation was tested for antibody reactivity by staining human or rat brain sections or HEK cells expressing NR1/NR2 heteromers of the NMDAR as previously described(Dalmau et al., 2007
; Dalmau et al., 2008
). Both patients' CSF and IgG decreased surface and total NMDARs to the same extent (Supplemental Fig. 1
Cell culture and patient antibody treatment
Briefly, isolated rat hippocampi were placed in Ca2+ free HBSS (Hanks balanced salt solution, Life Technology) containing 1% papain for 20 min., triturated in Basal Media Eagle (BME; Invitrogen) supplemented with B-27 (Life Technology) and plated at 100,000 or 400,000 (for biotinylation) cells per ml in Neural Basal (NB; Life Technologies) supplemented with 10% FBS (Hyclone), B-27, 1% Penicillin and Streptomycin (Life Technologies), and 1% L-Glutamine (Life Technologies) on poly-L-lysine coated (Sigma) coverslips in 24-well plates. Culture media was changed to NB supplemented with B27 at 4 div. Cells were maintained at 37 °C, 5% CO2,95% humidity; medium was changed weekly. Neurons were treated with CSF or IgG from individual patients or controls for 1 day beginning at 14 days in vitro; in some experiments, neurons were treated for 3 or 7 days beginning at 14 days in vitro.
Immunostaining for pre- and postsynaptic components, confocal imaging, and image analysis
To stain surface NMDAR clusters, control or treated neurons were washed in Neurobasal plus B27 and incubated with patient CSF containing anti-NR1 antibodies for 30 min., washed and incubated with fluorescently conjugated anti-human secondary antibodies for 30 min., and washed in PBS. Neurons were then fixed in 4% paraformaldehyde, 4% sucrose in PBS, pH = 7.4 for 15 min., permeabilized with cold 0.25% Triton X-100 for 5 min., and blocked in 5% normal goat serum (Invitrogen) for 1 hour at RT. Additional immunostaining was performed with various combinations of primary antibodies: to label glutamate receptors, anti-NR1 against the intracellular C-terminus (1:1000; Chemicon), anti-GluR1 (1:10; CalBioChem) or anti-GluR2 (1:100; Chemicon); to label postsynaptic densities, PSD-95 (1:500; Bioaffinity Reagents); to label dendrites, mouse anti-MAP2 (1:1000; gift from Dr. V. Lee); to label presynaptic terminals, mouse anti-SV2 (1:200; DHSB), guinea pig anti-VGLUT 1 (1:1000; Chemicon), or mouse anti-Bassoon (1:400; Stressgen Bioreagents). Antibodies were visualized after staining with the appropriate fluorescently conjugated secondary antibodies (1:200; Jackson ImmunoResearch).
Images were obtained using a confocal microscope (Leica TCS SP2). Images were thresholded automatically using iterative segmentation(Bergsman et al., 2006
), and the number and area of individual immunostained pre- or postsynaptic clusters were determined using interactive software (custom-written ImageJ macros). Clusters with pixel overlap of pre- and postsynaptic markers were considered colocalized and thus synaptic(Krivosheya et al., 2008
Biotinylation of surface proteins and analysis by Western blot
Neurons were treated with 1 μg − 1 mg/ml IgG for 1 day, washed with PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 (rinsing buffer) and incubated for 30 min. at 4 °C with 1 mg/ml Sulfo-NHS-Biotin (Thermo Scientific) in rinsing buffer. Neurons were then washed with rinsing buffer + 100 mM glycine (quenching buffer), incubated in quenching buffer for 30 minutes at 4°C to quench excess biotin, then lysed in RIPA buffer (150 mM NaCl, 1 mM EDTA, 100 mM Tris HCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4, supplemented with 1:500 protease inhibitor cocktail III, Calbiochem) at 4 °C for 1 hour. Lysates were cleared of debris by centrifugation at 12,400 × g for 20 min. An aliquot of the remaining supernatant was taken for the lysate fraction, and a second aliquot was incubated with avidin-linked agarose beads (Immobilized Monomeric Avidin, Thermo Scientific) overnight at 4 °C. After centrifugation, the supernatant was removed and the beads (surface fraction) were washed 1X RIPA buffer, 2X high-salt wash buffer (500 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.1% Triton X-100, pH 7.5), and 1X no-salt wash buffer (50 mM Tris, pH 7.5). The surface fraction was eluted from the beads with 2X sample buffer and proteins separated on an 8% gel using SDS-PAGE. Samples were transferred to nitrocellulose membranes and probed for antibodies against NR1 (1:1000, 556308, BD Pharmingen), NR2A (1:1000, AB1555, Millipore; 1:500, MAB5216, Millipore; 1:500, A6473, Invitrogen), NR2B (1:1000, AGC-003, Alomone; 1:500, 06–600, Upstate), GABAARα1 (1:1000, 06–868, Upstate), GABAARα2 (1:500, AB5984, Chemicon), GluR 2/3 (1:1000, 07–598, Upstate), PSD-95 (1:1000, 610496, BD Pharmingen), and actin (1:2000, A2066, Sigma). Actin and GABAARs were used as loading controls for total and surface fractions, respectively. Blots were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1:3000, Cell Signaling), and signals were visualized using chemiluminescence (SuperSignal Chemiluminescent Substrate, Thermo Scientific). All quantified films were in the linear range of exposure, were digitally scanned and signals quantified using NIH ImageJ.
Whole cell electrophysiological recordings of synaptic NMDA and AMPA receptor mediated currents
Whole cell voltage clamp recordings were performed as previously described(Elmariah et al., 2004
; Elmariah et al., 2005
) from 14 – 21 div pyramidal neurons treated for 24 hours with patient CSF containing anti-NR1 antibodies, control CSF or left untreated. Briefly, neurons were incubated in an extracellular physiological solution without Mg2+
(in mM: 119 NaCl, 5 KCl, 2 CaCl2
, 30 Glucose, 10 HEPES, pH = 7.4). Voltage-clamp recordings were made at RT (22–25 °C) using glass microelectrodes (resistance 4–6 MΩ) filled with a Cesium substituted intracellular solution (in mM: 100 Cesium gluconate, 0.2 EGTA, 5 MgCl2, 2 ATP, 0.3 GTP, 40 HEPES, pH = 7.2). Pipette voltage offset was neutralized before the formation of a gigaohm seal. Membrane resistance, series resistance, and membrane capacitance were determined from current transients elicited by a 5 mV depolarizing step from a holding potential of −80 mV, using the whole cell application of PatchMaster software (HEKA Elektronik). Criteria for cell inclusion in the data set included a series resistance ≤ 30 MΩ and stability throughout the recording period. Currents were amplified, low-pass filtered at 2.5 kHz, and sampled at 5 Hz using PatchMaster software. Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded at −70mV in the presence of TTX (1 μM) and picrotoxin (10 μM). APV (50 μM) and CNQX (10 μM) were bath applied to block NMDAR and AMPAR mediated currents respectively. mEPSC events were detected and analyzed using MiniAnalysis (Synaptosoft, Leonia, NY), which employs a threshold-based event-detection algorithm. NMDAR and AMPAR components of mEPSCs were separated temporally by their distinct kinetics(Hestrin et al., 1990
; Watt et al., 2000
; Yang et al., 2003
). The amplitude of the NMDAR mediated current was determined in a window between 15 and 25 ms after the peak of the AMPAR mediated component, which has a fast, < 1 ms rise time. All values are presented as mean ± s.e.m.
Fab fragment preparation and treatment
Fab fragments were prepared from serum IgG using a kit according to the manufacturer's directions (Fab preparation kit, Pierce Protein Research Products, Thermo Scientific). Briefly, serum IgG was digested for 2–4 hours at 37 °C with 1% (w/w) papain pH= 7.0 with 0.01 M cysteine, resulting in cleavage into Fab and Fc fragments. Fab fragments were isolated by chromatography and concentration determined by absorption at 280 nm, and then used to treat neurons at a concentration of 4 μg/ml. Control experiments showed that incubating neurons with patient Fab fragments for 30 min. resulted in surface staining of NR1 clusters (Supplemental Fig. 2
Alzet mini-pump placement, IgG infusion, and analysis of effects on NMDA receptors
7–8 week old female Lewis rats were anesthetized and a cannula was placed into the left hippocampus using predetermined coordinates (−3.2 mm posterior to bregma, 2 mm lateral, and 3 mm deep to the dura mater). The cannula was secured to a head probe mounted to the skull, and attached with sterile tubing to an Alzet minipump (Alzet brain infusion kit #3, pump model 2002) implanted subcutaneously on the back. Patient or control CSF was then delivered at a rate of 0.5 μl / hr for 2 weeks. Rats were then euthanized, brain tissue harvested, immersion fixed in 4% paraformaldehyde in PBS, pH = 7.4 for 15 min., cryoprotected in 30% sucrose in PBS, pH = 7.4 overnight at 4 °C, and snap frozen in isopentane cooled in dry ice. Frozen 10 μm sections from infused hippocampus (where the track of the cannula was visible) and contralateral matched area of the non-infused hippocampus were immunostained in parallel to determine the presence of human IgG and the levels of NR1 using the primary and secondary antibodies described above. The degree of cell death was assayed with TUNEL. Sections were imaged and thresholded with the same parameters, and confocally imaged and analyzed as described above.
Additionally, protein extracts from 20 μm sections of the infused and contralateral hippocampus were separated electrophoretically, transferred to nitrocellulose, incubated with anti-NR1 antibody (Chemicon), and the amount of NR1 protein quantified as described above, using Tubulin as a loading control.
Immunostaining, imaging, and image analysis of human tissue
Hippocampal sections of human tissue were immunostained in parallel as described above. Control and patients' tissue sections were imaged with a Zeiss Axioskop 2 plus (software AxioVision 4.5) with identical optical settings and exposure times. For analysis of high magnification regions 7–10 images were collected from the CA1 region of the hippocampus. These images were inverted and a cumulative histogram of pixel intensity was calculated for each image. The average cumulative histogram of pixel intensity was generated for each sample and the cumulative probability of pixel intensity for each sample was determined, plotted and compared using a paired Komolgorov-Smirnov test (see below).
Titer dependence was assessed with a linear regression analysis. In experiments involving two conditions, the data was analyzed with a two-tailed unpaired Student's t test. In experiments involving three or more conditions, the normality of the data was analyzed with the D'Agostino and Pearson omnibus normality test, before using a one-way ANOVA test followed by Bonferroni's multiple comparison test. Differences in distributions of NR1 intensity were assessed with a paired Komolgorov-Smirnov test. All values are presented as mean ± s.e.m.