Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus

Hippocampal Lamination Defects in ITSN1 KO Mice.

To explore the physiological functions of ITSN1 in vivo, we analyzed the overall brain architecture of KO mice deficient in ITSN1 (25, 26). Nissl-stained sections of hippocampi from ITSN1 KO mice showed a gross dispersion of pyramidal neurons in the CA1 and CA3 areas and an altered shape of the dentate gyrus (DG) (Fig. 1A), suggesting a possible defect in neuronal migration. A similar phenotype has been observed in KO mice lacking Reelin receptors (e.g., VLDLR, ApoER2) (13). Defects in Reelin signaling are accompanied by malformation of the radial glial scaffold in the DG (17, 18); therefore, we examined whether loss of ITSN1 affects radial glial cell morphology. Indeed, in KO mice lacking ITSN1, radial glial processes immunopositive for the radial glial marker Nestin (Table S1) were severely disorganized (Fig. 1B), and their numbers were reduced compared with those in wild-type (WT) littermates (Fig. 1 B and C). The total volume of the DG was unaffected by loss of ITSN1 (Fig. S2 A and B). A similar disorganization of the radial glial scaffold (18) and reduction in the number of radial glial processes was observed in ApoER2 KO mice (Genetic Interaction of ITSN1 with Reelin Signaling). A key function of the radial glial scaffold is to guide neuronal migration in the DG to allow the integration of newborn neurons into the neuronal network and enable learning and memory in adults (2, 6, 7). Conditional defects in Reelin signaling impair radial neuronal migration, resulting in the ectopic location of newborn neurons in the hilar region of the DG (20). A similar accumulation of ectopic newborn doublecortin (Dcx)-positive neurons in the hilus was found in ITSN1 KO mice (Fig. 1 D and E). These data suggest that ITSN1 is required for normal hippocampal cell layering, formation of the radial glial scaffold, and proper migration of newborn neurons in the hippocampus.

In contrast, we did not detect consistent defects in our ITSN1 KO mice with respect to cortical midline connectivity (Fig. S2 C and D) that were previously reported in a gene trap line with different genetic background (35). Moreover, loss of ITSN1 did not affect the kinetics of presynaptic vesicle exocytosis or endocytic membrane retrieval as measured by fluorescence analysis of pHluorin-tagged synaptic vesicle proteins (Fig. S3 A and B), levels of exocytic/endocytic proteins (Fig. S3C), and the number, area, length, and morphology of dendritic spines (Fig. S3 D–H).

Intersectin Is Required for Reelin Signaling to Facilitate NMDAR-Mediated LTP.

Given that pyramidal cell dispersion, malformation of the radial glial scaffold, and defects in neuronal migration in the hippocampus have been observed in mouse mutants of the Reelin pathway (2, 6, 7, 13, 20) we hypothesized that ITSN1 may constitute a component of Reelin signaling. To directly test this idea, we probed the ability of Reelin to augment hippocampal long-term potentiation (LTP), a process that strictly depends on signaling via the ApoER2 and VLDLR (22, 23). Brief theta-burst stimulation (TBS) of CA1 Schaffer collaterals from WT mice perfused with control media induced reliable LTP that was strongly facilitated by application of Reelin (Fig. 2 A and C), in agreement with previously reported data (22, 23). Application of Reelin-containing media had no effect on baseline transmission (Fig. S4C). In contrast, Reelin failed to augment TBS-induced LTP in hippocampal slices from ITSN1 KO mice (Fig. 2 B and C). Loss of ITSN1 did not affect TBS-induced LTP as such (Fig. 2 B and C and Fig. S5C) or basal parameters of synaptic transmission (Fig. S5 A and B), in agreement with previous reports (25, 35).

To further investigate the mechanism underlying Reelin-mediated augmentation of LTP, we analyzed the effect of Reelin on N-methyl-d-aspartate receptor (NMDAR) potentials, late components of TBS-elicited field excitatory postsynaptic potentials (fEPSPs) (Fig. S4 A and B). In agreement with previous data (22, 23), Reelin application potentiated NMDAR-mediated potentials in hippocampal slices from WT mice (Fig. 2 D and E). In contrast, ITSN1 KO mutants showed no elevation of NMDAR-mediated potentials above control levels (Fig. 2 D and F). α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated potentials, measured as the first response during TBS, were not affected in Reelin-perfused slices (Fig. S4 A and D). As a result, the NMDA/AMPA ratios were facilitated in WT slices, but not in slices from ITSN1 KO mice (Fig. S4 E and F).

To probe whether ITSN1 is selectively required for Reelin-dependent facilitation of hippocampal LTP, we induced LTP in the presence of brain-derived neurotrophic factor (BDNF), another well-established facilitator of LTP (36). Recordings in the presence of BDNF revealed that LTP was facilitated in both WT and ITSN1 KO mice to the same level (Fig. S5 D and F), indicating that ITSN1 is dispensable for BDNF-dependent LTP augmentation. To study a possible role of ITSN1 for hippocampal plasticity in general, we chemically induced synaptic plasticity by applying the voltage-dependent K⁺ channel blocker tetraethylammonium (TEA). Incubation of slices with TEA induces spontaneous discharges of neurons and thereby a voltage-gated Ca²⁺ channel-dependent form of LTP (37). TEA equally facilitated synaptic responses in both genotypes to the same extent (Fig. S5 E and F). Collectively, these data demonstrate that ITSN1 is specifically required for Reelin signaling to augment NMDAR-mediated hippocampal synaptic plasticity, whereas loss of ITSN1 phenotypically mimics KO mice lacking the Reelin receptors ApoER2 or VLDLR or their signaling adaptor Dab1.

ITSN1 associates with the VLDLR and its downstream signaling adaptor Dab1 and facilitates Reelin-induced Dab1 phosphorylation.

To obtain molecular insights into the mechanism by which ITSN1 mediates Reelin signaling we probed for direct physical interactions between ITSN1 and components of the Reelin pathway. We found that ITSN1 can associate with the VLDLR, but did not detect reliable binding to ApoER2 (Fig. 3A). ITSN1-VLDLR complex formation involved the SH3 domains of ITSN1 and the intracellular domain of VLDLR (Fig. 3B). Furthermore, ITSN1 also bound to the ApoER2/VLDLR-associated adaptor Dab1 (Fig. 3C), a key signaling factor that transmits Reelin signals to intracellular effectors by undergoing tyrosine phosphorylation (17, 20, 38).

Given that ITSN1 can associate with VLDLR and Dab1, we hypothesized that such a scaffolding function of ITSN1 may facilitate Reelin-induced phosphorylation of Dab1, a key step in the Reelin signaling cascade. We cultured neurons from control and ITSN1 KO mouse embryos (E16) and acutely treated them with Reelin. Whereas Reelin application led to a robust increase in Dab1 phosphorylation in WT neurons, Dab1 phosphorylation was significantly reduced in neurons from ITSN1 KO embryos (Fig. 3D). In line with this acute defect in Reelin signaling in cultured neurons, we found the significantly reduced steady-state levels of phospho-Dab1, which reflect constitutive Reelin signaling, in hippocampal lysates from ITSN1 KO mice (Fig. 3E). Total Dab1 levels remained unaltered in hippocampal lysates and cortical embryonic neuron cultures from ITSN1-deficient animals (Fig. S6 A and B). We did not observe any reduction in Reelin, ApoER2, or VLDLR levels (Fig. S6 A and C), suggesting that defective Reelin signaling in the absence of ITSN1 is not a consequence of impaired Reelin production or reduced receptor levels. Instead, we found increased total levels of Reelin in the hippocampi of ITSN1 KO mice (Fig. S6 C and D), likely as a result of compensatory adaptation.

The foregoing results corroborate a role for ITSN1 in Reelin signaling. They further indicate that ITSN1 acts as a molecular scaffold that links VLDLR to Dab1 to facilitate Dab1 phosphorylation downstream of Reelin binding, thereby identifying ITSN1 as a regulator of the Reelin pathway that may act predominantly via the VLDLR-Dab1 axis.

Genetic interaction of ITSN1 with Reelin signaling.

If ITSN1 were a component of Reelin signaling associated with VLDLR, then ITSN1 would be expected to genetically interact with other Reelin signaling components, particularly with ApoER2 in vivo. To test this hypothesis, we crossed KO mice lacking ITSN1 with mice deleted for ApoER2. Loss of ApoER2 or VLDLR has previously been shown to cause a weak Reeler-like phenotype that is enhanced in double-KO (DKO) mice lacking both Reelin receptors (13). In histological sections from WT, ITSN1 KO, and ApoER2 KO mice, layer 1 of the neocortex was largely devoid of cell bodies, confirming previously reported findings (13). In contrast, ITSN1/ApoER2 DKO mice displayed pronounced abnormal neuronal migration into cortical layer 1 (Fig. 4A). This defect was accompanied by the near-complete absence of the normal radial arrangement of MAP2-containing dendritic processes of cortical neurons in ITSN1/ApoER2 DKO mice (Fig. 4B) similar to, albeit less severe than, the phenotype of VLDLR/ApoER2 DKO mice (13).

To further probe the genetic interaction between ITSN1 and ApoER2, we analyzed hippocampal cell layering. We confirmed the previously observed dispersion of hippocampal pyramidal neurons in ApoER2 KO mice, e.g., CA1 splitting into two layers (13). DKO of both ApoER2 and ITSN1 exacerbated neuronal dispersion in the hippocampus (Fig. 4C), with the CA1 region split into three partially dispersed layers (Fig. 4C, Upper, Insets), whereas neurons in CA3 (Fig. 4C, Lower, Insets) were widely scattered. Moreover, paired loss of both ApoER2 and ITSN1 resulted in a significant further reduction in the number of radial glial cell processes in the hippocampus (Fig. 4 D and E) and in a further increase in ectopic Dcx-positive neurons in the hilar region of the DG (Fig. 4 F and G). In contrast, no further enhancement of hippocampal cell dispersion was observed in ITSN1/VLDLR DKO mice (Fig. S7A), providing further support to the idea that ITSN1 predominantly regulates the VLDLR-Dab1 branch of the Reelin signaling pathway. We conclude that ITSN1 and ApoER2 interact genetically in vivo, with their combined loss leading to cortical and hippocampal malformations similar to, although slightly less severe than, those observed in Reeler or VLDLR/ApoER2 DKO mice. Collectively, our data demonstrate that ITSN1 physically, functionally, and genetically interacts with Reelin receptors and thus represents a hitherto unknown component of the Reelin pathway that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration and synaptic plasticity.

Maintenance and Breeding of Mouse Strains.

The ITSN1 KO mice were a kind gift from Melanie Pritchard, Monash University, Melbourne, Australia. Heterozygous mice were interbred to yield ITSN1 KO mice and corresponding WT control animals. ApoER2 and VLDLR KO mice were generously provided by Joachim Herz, University of Texas Southwestern Medical Center, Dallas. To obtain ITSN1/ApoER2 or ITSN1/VLDLR DKO mice, ITSN1 KO mice were interbred with ApoER2 or VLDLR heterozygous mice. The resulting double-heterozygous mice were bred to produce DKO and single-KO mice, as well as control animals (single- or double-heterozygous mice). Genotypes were determined by PCR analysis from genomic DNA obtained from tail, toe, or ear biopsy specimens before the experiments. All animal experiments were approved by the Regional Office for Health and Social Affairs Berlin.

Immunohistochemical Analysis.

Immunohistochemistry was performed on free-floating 40-µm-thick brain sections of paraformaldehyde (PFA)-perfused mice using specific antibodies as described previously (46). Immunofluorescence images were acquired using a Zeiss LSM 710 or 780 confocal microscope. Numbers of processes or cells were counted using ImageJ software.

GST Pull-Down Assays and Detection of Phosphorylated Dab1.

GST-tagged proteins were recombinantly expressed in Escherichia coli (BL-21) and purified using GST•Bind Resin (Novagen). For this, 100 µg of purified GST-fusion proteins were incubated with cell or brain extract for 2–4 h at 4 °C on a rotating wheel. Bound proteins were eluted by SDS sample buffer. Dab1 was immunoprecipitated from hippocampal tissue extracts in the presence of Phosphatase Inhibitor Mixture 2 and 3 (Sigma-Aldrich). Protein A/G PLUS agarose beads (Santa Cruz Biotechnology) were loaded with 1 µL of antibody per 500 µg of extract, and antibody-coupled beads were incubated with ultracentrifuged extract for 2 h at 4 °C. Levels of phosphorylated Dab1 were detected by SDS/PAGE and immunoblotting with the phosphotyrosine-specific antibody 4G10 (EMD Millipore).

Electrophysiology.

Acute hippocampal field electrophysiology was performed using 2- to 3-mo-old WT and ITSN1 KO mice. Mice were decapitated, and brains were quickly sectioned into 350-μm-thick sagittal slices and then transferred to recover in a resting chamber (22–24 °C) filled with artificial cerebrospinal fluid (ACSF) containing 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 24 mM NaHCO₃, 1.5 mM MgSO₄, 2 mM CaCl₂, and 25 mM glucose (pH 7.35–7.4). After recovery, slices were transferred to a submerged recording chamber supplied with continuously bubbled ACSF with solution exchange of 3–5 mL/min at room temperature (22–24 °C). CA3–CA1 Schaffer collaterals were stimulated in stratum radiatum using a glass electrode filled with ACSF (1–1.5 MΩ), and a similar glass electrode (1.5–2.5 MΩ) placed in the distal part of the CA1 region was used to record fEPSPs. Electrical pulses of 0.2 ms were delivered for stimulation of Schaffer collaterals, and basal excitatory synaptic transmission was evaluated using input-output stimulus response curves. Short-term plasticity of synaptic transmission was tested using a paired pulse facilitation (PPF) protocol, in which two pulses were delivered at various interpulse intervals (10–500 ms) and the facilitation was calculated as the ratio of the amplitude of the second response compared with the first. To test synaptic plasticity, we induced LTP using single TBS comprising eight bursts delivered every 200 ms, with each burst containing four pulses at 100 Hz. The data were recorded at a sampling rate of 10 kHz, low-pass filtered at 3 kHz, and analyzed using PatchMaster software and an EPC 9 amplifier (HEKA Elektronik).

Statistical Analysis.

The data in graphs are mean ± SEM values from several experiments/mice (n and N, respectively). For comparison of WT and ITSN1 KO littermates, statistical analysis was performed using the paired two-tailed t test, unless indicated otherwise. For comparison of more than two groups, one-way ANOVA with Student–Newman–Keuls (SNK) post hoc analysis was performed.

SigmaPlot (Systat Software) was used for electrophysiological data analysis, statistical evaluation, and data presentation. Data in the figures are presented as mean ± SEM, and the number of tested slices (n) and mice (N) are indicated in the legends. For statistical evaluation, the two-tailed unpaired Student´s t test or repeated-measures (RM) ANOVA (significant difference depicted over a line encompassing the curve) was used, with the factor genotype or treatment. Significant main effects of treatments detected by two-way RM ANOVA were further explored using SNK post hoc analysis. Sex was not included as a factor in the analyses, because the data did not show any sex-related alterations. In the figures, *P < 0.05, **P < 0.01, statistically significant difference between control medium and Reelin-treated WT slices; ##P < 0.01, significant difference between Reelin-treated WT and ITSN1 KO slices.

Detailed information on plasmids; neuronal cell culture, transfection, and immunostaining; histology and DG volume estimation; tissue and cell extracts; crude synaptic vesicle (LP2) preparation; Cdc42 activity assay (G-LISA); surface biotinylation of hippocampal cultures; endocytosis assays; growth cone collapse assay; and Reelin production, concentration, and application to neuronal cultures is available in SI Materials and Methods.

Plasmids.

The expression construct for murine ApoER2 was a kind gift from G. William Rebeck, Georgetown University, Washington, DC. For mammalian expression, coding sequences were combined with an N-terminal EGFP tag and a C-terminal HA tag. For bacterial expression, the intracellular region of the ApoER2 (ApoER2-ICR: amino acids 756–870) and VLDLR (VLDLR-ICR: amino acids 792–845) were cloned into pGEX4T-1. GST-tagged domains of human ITSN1 in pGEX4T-1 have been described previously. cDNA encoding human INTS2 SH3A-C was cloned into pGEX4T-1, similar to ITSN1 constructs. Murine Dab1 and Dab2 constructs, kindly provided by Jonathan Cooper, Fred Hutchinson Cancer Center, Seattle, were cloned into pcDNA3.1 with an N-terminal Myc-tag. HA-tagged mouse EphB1 in pCAG was a generous gift from Carol Mason, Columbia University, New York. The coding sequence for ephrinB2 was amplified from murine cDNA and cloned into pEGFP-N1.

Neuronal Cell Culture from Postnatal Brains, Transfection, and Immunostaining.

Primary hippocampal neurons (p1–p4 mice) were prepared in mass and cultured in Neurobasal-A/B27, GlutaMAX, and penicillin-streptomycin. Hippocampi were pooled from several genotypically identical littermates. Cells were transfected by calcium phosphate transfection (Promega). Cultured cells were fixed using 4% (wt/vol) PFA and 4% sucrose in PBS for 15 min at room temperature. Fixed cells were blocked and permeabilized with PBS containing 10% goat serum and 0.3% Triton X-100 for 30 min, followed by incubation with primary antibodies in 0.3% Triton X-100 in PBS for 1 h. Secondary antibodies (coupled to Alexa Fluor 488, 568, and 647; Life Technologies) were applied for 30 min in PBS plus 0.3% Triton X-100. To detect the surface pool of plasma membrane proteins, the protocol was carried out in the absence of Triton X-100.

Reelin Production and Concentration.

CER cells, from Tom Curran, University of Pennsylvania and Children's Hospital of Philadelphia, Philadelphia (for electrophysiology), or HEK293 cells, from H.B. (for neuron cultures), stably expressing full-length Reelin, were cultured in four 15-cm dishes in DMEM with 10% FBS and penicillin-streptomycin. To obtain Reelin, 80–100% confluent 15-cm dishes were washed meticulously with PBS three times, and cells were incubated with starvation medium (DMEM containing 20 mM Hepes, pH 7.4, but no serum and antibiotics) for 24 h. The cell supernatant was harvested, and new starvation medium was added for another 24 h. To enrich Reelin, the combined supernatants were first precleared by centrifugation at 1,000 × g for 5 min and then concentrated ∼20 times using an Amicon Ultra 15-mL 100-kDa cutoff centrifugal filter (EMD Millipore). Protein concentration was measured by the Bradford assay, and the Reelin content was verified by a 4% Coomassie-stained SDS/PAGE gel, after which the concentrate was frozen in liquid nitrogen and stored at −80 °C until use. Concentrated supernatant from CEP4 cells (for electrophysiology, from Tom Curran) or HEK293 cells (for neuron cultures, from H.B.) served as a negative control.

Culture of E16.5 Neurons, Reelin Treatment, and Phospho-Dab1 Detection.

At E16.5, the uteri of pregnant females from timed matings of ITSN1 heterozygous mice were transferred into PBS. After an additional wash step in PBS, the embryos were extracted and placed singly into fresh dishes with ice-cold PBS. The heads were cut off and transferred into a dish with ice-cold HBSS, and parts of the bodies were processed for genotyping. The cortices were dissected, cut into 15–20 small pieces, and transferred with a pipette into an Eppendorf tube. When the pieces had settled to the ground, the solution was exchanged to 500 µL of cold 0.1% trypsin in HBSS, and the cortices were placed on a rotating wheel at 37 °C for 15 min for trypsinization. The samples were then placed on ice, and 200 µL of ice-cold soybean trypsin inhibitor (STI) solution (0.4 mg/mL STI, 0.25 mg/mL DNase, 0.3% BSA, and 12 mM MgSO₄ in HBSS) was added, followed by centrifugation at 200 × g for 4 min.

After washing the pellet once in 700 µL of HBSS and centrifugation as above, the tissue pieces were resuspended in 400 µL of STI solution and dissociated by pipetting 10 times with a 1-mL pipette with a 10-µL pipette tip on top of the 1-mL pipette tip. The cells were then centrifuged for 8 min at 300 × g, resuspended in 1 mL of neuron growth medium (2% B27 supplement, 1 mM l-glutamine, and 1× penicillin-streptomycin in neurobasal medium), and counted. Between 9 and 13 million cells were obtained per embryo, and 4.5 million cells per well were seeded in 1.5-mL neuron growth medium into two six-well plates coated with 500 µL of 15 µg/mL poly-l-lysine for 2 h at 37 °C, washed once with sterile water, and left to dry. The cells were cultured at 37 °C in 5% CO₂. After 2 h, an additional 1.5 mL of neuron growth medium was added. On day in vitro (DIV) 4, 1.5 mL of the neuron growth medium was exchanged for fresh neuron growth medium. On DIV5, 1 mL of neuron growth medium per well was collected and then centrifuged at 800 × g for 5 min. After either control medium or Reelin-containing medium was diluted by 1:6 in the collected medium, the medium remaining on the cells was replaced by 800 µL of this mixture. Following a 20-min incubation at 37 °C, the cells were gently washed with 2 mL of ice-cold PBS containing 10 mM MgCl₂. Immediately afterward, the PBS was replaced by 200 µL of lysis buffer [20 mM Hepes, pH 7.4, KOH, 100 mM KCl, 2 mM MgCl₂, 1% Triton-X-100, 5 mM EDTA, 2 mM EGTA, 1 mM PMSF, 5 µL/mL mammalian inhibitor mixture (Sigma-Aldrich), and 10 µL/mL phosphatase inhibitor mixture 2 and 3 (Sigma-Aldrich)]. After 30 min of lysis on ice, the lysate was transferred into Eppendorf tubes, and left for another 5 min on ice, during which it was vortexed three times before centrifugation at 17,000 × g for 10 min.

The protein concentration was determined, and equal amounts of protein for the control and KO samples to be compared (139–225 µg) were incubated for 2 h at 4 °C in 200 µL of lysis buffer with antibody coupled to protein A/G Sepharose beads. Immunoprecipitation was performed using either 1 µL of anti-Dab1 antibody (EMD Millipore, AB5840, which became unavailable at some point) or 2 µL of anti-Dab1 antibody (Rockland/Biomol; 100-401-225S). After three washing steps with Triton X-100–containing lysis buffer and one washing step with Triton X-100–free lysis buffer, the proteins were eluted in 40 µL of sample buffer for 5 min at 95 °C. The entire 40 µL per sample was separated on an 8% SDS/PAGE gel, blotted onto nitrocellulose membrane, and detected first with anti-phosphotyrosine antibody (1:1,000) and then with anti-Dab1 antibody (EMD Millipore, 1:1,000 or Rockland, 1:2,000). HRP-coupled secondary antibodies were used together with picoECL reagent, and the signals were detected using the Bio-Rad ChemiDoc MP Imaging System. Band intensities were quantified by ImageJ, version 2.0.0-rc-59/1.51j, using the “analyze gels” function. The increase in signal intensity after the addition of Reelin was calculated for each genotype.

Histology and DG Volume Estimation.

Animals were perfused with 4% PFA, and their brains were isolated and cryoprotected with 20% glycerol and 2% DMSO in 125 mM phosphate buffer, pH 7.4. A standard Nissl staining protocol was used to assess general anatomic features. For DG volume determination, every sixth section was stained with the Nissl method. Images from all sections containing the DG were recorded, the DG was outlined, and the area was measured using ImageJ software. Volume was estimated using the formula ∑A × h × n, where ∑A is the sum of the area measured on each section, h is the section thickness (40 µm), and n is the section interval. Golgi impregnation of brains was performed with the FD Rapid GolgiStain Kit (FD NeuroTechnologies) according to the manufacturer’s instructions.

Tissue and Cell Extracts.

Tissue was homogenized in lysis buffer [100 mM KCl, 2 mM MgCl₂, 20 mM Hepes, pH 7.4, 1% Triton X-100, 1 mM PMSF, and 0.3% mammalian protease inhibitor mixture (Sigma-Aldrich)] using a homogenizer pestle. HEK293 cells were transfected with calcium phosphate or JetPRIME (Polyplus Transfection) and harvested in lysis buffer. Tissues/cells were incubated for 30 min at 4 °C and then centrifuged at 17,000 × g for 10 min at 4 °C. For interaction studies, lysates were further cleared by ultracentrifugation at 180,000 × g for 20 min at 4 °C. Protein samples were diluted in SDS sample buffer, resolved by SDS/PAGE, and processed for immunoblotting. Samples were analyzed by SDS/PAGE and immunoblotting. Bound primary antibodies were detected by incubation with IRDye 800CW-conjugated secondary antibodies and the Odyssey Fc Imaging system (LI-COR Biosciences). Alternatively, HRP-conjugated secondary antibodies and ECL Substrate (LI-COR; P/N 926–80100) were used. Chemiluminescence was detected with the Bio-Rad ChemiDoc MP Imaging System. The antibodies used in this study are listed in Table S1.

Crude Synaptic Vesicle (LP2) Preparation.

For subcellular fractionation of brain tissue, a mouse brain was homogenized in 12 mL of ice-cold homogenization buffer with 20 strokes at 900 rpm. After centrifugation for 10 min at 800 × g, the supernatant (S1) was centrifuged again, for 15 min at 10,000 × g. The pellet (P2) was resuspended in 12 mL of ice-cold homogenization buffer and then centrifuged at 10,000 × g for 15 min. The resulting pellet (P2′) was resuspended in 1.2 mL of homogenization buffer, and synaptosomes were lysed osmotically by the addition of 9 mL of ice-cold MilliQ H₂O and homogenization with three strokes at 2,000 rpm. The solution was centrifuged at 25,000 × g for 20 min, and the resulting supernatant (LS1) was centrifuged at 200,000 × g for 2 h. The pellet containing crude synaptic vesicles (LP2) was resuspended in 50 µL of homogenization buffer and stored in 1× SDS sample buffer.

Cdc42 Activity Assay (G-LISA).

For quantification of active Cdc42 the G-LISA activation assay from Cytoskeleton, Inc. was performed according to the manufacturer's instructions.

Surface Biotinylation of Hippocampal Cultures.

Cells were incubated with EZ-Link Sulfo-NHS-SS-biotin solution (0.5 mg/mL in PBS²⁺ containing 1 mM MgCl₂ and 1 mM CaCl₂; Thermo Fisher Scientific) for 15 min at 4 °C. Residual biotin was quenched with glycine (50 mM in PBS²⁺). Cell lysates were incubated with 100 µL of streptavidin beads for 2 h at 4 °C. Surface proteins were eluted with SDS sample buffer and detected by SDS/PAGE and immunoblotting.

Endocytosis Assays.

Antibody uptake experiments were performed with DIV7 hippocampal neurons, transfected on DIV3 with EGFP-VLDLR-HA or EGFP-ApoER2-HA. Transfected cells were incubated with anti-EGFP antibody (1:500; Abcam) for 15 or 30 min at 37 °C or for 30 min at 8 °C (control). Cells were fixed, and the surface-bound antibody pool was stained with goat anti-rabbit Alexa Fluor 647 (Invitrogen) before postfixation with 2% PFA for 10 min. Neurons were permeabilized, and internalized antibodies were stained with goat anti-rabbit Alexa Fluor 568 (Invitrogen). For uptake assays with preclustered ephrinB-Fc, ephrinB1-Fc and ephrinB2-Fc chimeric proteins were preclustered with goat anti-human Fc antibodies at a 2:1 molar ratio for 1 h at room temperature in PBS. Uptake of ligand (final concentration 500 ng/mL) was allowed for 15 min at 37 °C. Surface-bound ephrinB-Fc was stained with donkey anti-goat Alexa Fluor 488 before postfixation with 2% PFA for 15 min. After permeabilization, internal ephrinB-Fc was stained with donkey anti-goat Alexa Fluor 568. To assay transendocytosis of ephrinB/EphB complexes, hippocampal glia cultures were prepared similarly to hippocampal neurons, but cultured in DMEM with 10% FBS and penicillin-streptomycin. Cells were isolated and cultured for two or three passages before transfection with Lipofectamine 2000. Two dishes each of WT and ITSN1 KO cells were transfected with HA-tagged EphB1 and GFP-tagged ephrinB2, respectively. The next day, WT cells, transfected with HA-EphB1, and ITSN1 KO cells, transfected with ephrinB2-GFP, were split and mixed in a 1:1 ratio. The same was done with WT cells, transfected with ephrinB2-GFP, and ITSN1 KO cells, transfected with HA-EphB1. After 1 d, cells were fixed and stained for HA and GFP. Coverslips were examined for transendocytosis between neighboring transfected cells of different origin.

Growth Cone Collapse Assay.

Hippocampal explants were isolated from embryonic mice (E16–E18). The embryonic brains were isolated and stored in ice-cold HBSS while genotyping was carried out. Hippocampi were chopped into 500-µm-thick pieces using a McIlwain tissue chopper (Mickle Laboratory Engineering), and hippocampal tissue was plated on poly-l-lysine/laminin (100 µg/mL and 20 µg/mL)-coated coverslips. Chimeric murine ephrinB2/human Fc region (R&D Systems) was preclustered with a rabbit anti-human IgG Fc secondary antibody at a molar ratio of 1:2 for 1 h at room temperature and used at a final concentration of 2.5 µg/mL Preclustered ephrinB2/Fc or control antibody were added to hippocampal explants at DIV1 or DIV2 and incubated for 60 min at 37 °C. For fixation of explant cultures, 4% PFA with 20% sucrose was added on top of the culture medium.

Electrophysiological Recordings.

Mice were decapitated after cervical dislocation, and brains were quickly extracted into ice-cold dissection ACSF containing 2.5 mM KCl, 1.25 mM NaH₂PO₄, 24 mM NaHCO₃, 1.5 mM MgSO₄, 2 mM CaCl₂, 25 mM glucose, and 250 mM sucrose. The brains were dissected into equal hemispheres, glued onto the object mounting platform with their cut surface, and sectioned into 350-μm-thick sagittal slices using a vibroslicer (VT 1200S; Leica). Slices were prepared in preoxygenated and continuously bubbled (95% O₂/5% CO₂) dissection ACSF at low temperature. The slices with clearly visible hippocampus were collected in a resting chamber containing ACSF in which 250 mM sucrose was replaced with 120 mM NaCl (pH 7.35–7.4) and then incubated for at least 1.5 h to allow recovery before recordings. After recovery, the slices were transferred to a submerged chamber (RC-27L; Warner Instruments) for recording. The recording chamber was supplied with continuously bubbled ACSF with a solution exchange of 3–5 mL/min at room temperature (22–24 °C). An upright microscope (BX61WI; Olympus) was used for the positioning of the slices to provide access to the CA1 region of the hippocampus and electrode placement. CA3–CA1 Schaffer collaterals were stimulated in the stratum radiatum using a glass electrode filled with ACSF (1–1.5 MΩ), and a similar glass electrode (1.5–2.5 MΩ) placed in the distal part of the CA1 region was used for fEPSP recordings. Glass electrodes were prepared from 1.5-mm glass capillaries (Hilgenberg) using a micropipette puller (P-1000; Sutter Instruments). Baseline stimulation of 0.2-ms electrical pulses were delivered every 20 s at the stimulation intensity that induced ∼30–50% of the maximal responses, to monitor the stability of recordings.

After at least 10 min of stable baseline recordings, basal excitatory synaptic transmission was tested using input-output stimulus response curves. The stimulation intensity was increased in 20-µA steps until maximal fEPSP amplitudes, defined as responses with a superimposed PS component on fEPSPs, were obtained. The fEPSP amplitudes were plotted vs. fiber volley amplitudes to estimate basal synaptic function (Fig. S5A). The short-term plasticity of synaptic transmission was tested using a PPF protocol in which two pulses were delivered at various time intervals (10, 20, 50, 100, 200, and 500 ms), and the facilitation was calculated as a ratio of the amplitude of the second response compared with the first (Fig. S5B). The stimulation intensity during PPF was set to induce one-third of the maximal responses, and for short intervals (10 and 20 ms), the first fEPSPs were digitally subtracted before measurements of the second fEPSPs. Each value measured is an average of five consecutive stimulations repeated every 20 s for stimulus responses and every 30 s for PPF measurements.

To test synaptic plasticity, LTP was induced using single TBS delivered at stimulation intensity, which induced 50% of the maximal responses. LTP was calculated as facilitation of mean slope of the fEPSPs at 50–60 min after TBS compared with 0–10 min before TBS application, which was set at 100%. The TBS contained eight bursts delivered every 200 ms, with each burst containing four pulses delivered at 100 Hz. For LTP experiments, basal stimulation was delivered every 20 s; for data presentation, each value is an average of three consecutive time points, with the delivery of TBS indicated by an arrow.

To study the effect of Reelin on synaptic plasticity, slices were acutely perfused with Reelin (5 µg/mL total protein from cell culture supernatants, of which only a fraction was Reelin) for 20 min. Reelin was applied 15 min before and 5 min after TBS. No effect of Reelin-containing medium on basal synaptic transmission on baseline responses recorded before LTP induction (Fig. S4C) and the first AMPA responses measured during TBS (Fig. S4D) was observed. Amplitudes of the first AMPA and the late NMDAR-mediated responses during TBS in the presence or the absence of control media and/or Reelin were analyzed. The measurements of the first AMPA-mediated responses is indicated by the arrow in Fig. S4A, and horizontal lines indicate the measurements of the late NMDAR-mediated responses (80–180 ms after the beginning of each theta burst) in which the mean amplitude of the late NMDAR-mediated response were measured (Fig. 2D and Fig. S4A). AMPA/kainate receptor antagonist NBQX (10 µM; Tocris) was used to isolate NMDA receptor-mediated responses and demonstrate that AMPA-mediated responses ended when NMDAR-mediated responses were measured (Fig. S4B). For some experiments (Fig. S5E), the voltage-dependent K⁺ channel blocker TEA (25 mM; Sigma-Aldrich) was applied to induce LTP chemically. Human recombinant BDNF (Peprotech) was used as an alternative plasticity facilitator to test the specificity of ITSN1 for Reelin signaling. BDNF (20 ng/mL) was applied at least 30 min before LTP induction, with special care taken to solve and wash BDNF into the slices. The recording system was washed with 0.1% BSA before BDNF application to minimize protein precipitation to the perfusion system.