Fig 2: Model of ApoER2 and VLDLR mediated signaling during cortical development. Radial glia cells divide asymmetrically to produce postmitotic neurons. During early stages of cortical development these neurons migrate via somal translocation. When the thickness of the cerebral wall increases neurons switch their migration mode to multipolar migration followed by locomotion. Thus, polarized neurons leave the ventricular (VZ) and subventricular zone (SVZ), depolarize and migrate through the IZ via a multipolar migration mode. The depolarization step is ill defined and may involve ApoER2 (A)-mediated signaling driven by the central Reelin fragment R3–6 (box 1). Repolarization of the neurons as prerequisite to glia cell guided locomotion is induced by Dab1 phosphorylation mediated by a netrin 1—deleted in colon cancer (DCC) signaling cascade and/or by the canonical Reelin pathway (box 2). Locomotion and terminal translocation is orchestrated by a complex set of Reelin (full length, FL) signals transmitted by clustering of ApoER2 (A) and VLDLR (V) homo- oligomers, leading to Dab1 phosphorylation (box 3). CP, cortical plate; IZ, intermediate zone; MZ, marginal zone.

Fig 3: Dab1 phosphorylation depends on EGFR kinase activity. Western blot analysis of protein extracts from HEK293 (A) and NIH3T3 (B) cells expressing EGFR and Dab1. Inhibitors of EGFR phosphorylation, Gefitinib (Lane 3) and Neratinib (Lane 4) were added 2 h before EGF treatment (3 min at 37 °C). Lane 1 represents untreated negative controls, Lane 2 represents the EGF treated positive controls. Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). (C) Western blot analysis of protein extracts HEK293T expressing Dab1 and either a wild type (WT)-like EGFR or a kinase-dead (KD) EGFR. Lanes 1 and 3 represent the untreated negative controls and Lanes 2 and 4 represent EGF treatment. Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Bands were scanned with ChemiDoc Touch Imaging System (BioRad, Hercules, CA, USA) and the anti-phosphotyrosine signal was normalized to the anti-Dab1 band. The level of Dab1 phosphorylation in the untreated WT EGFR (lane 1) control was set to 1. Relative intensity is shown in a box above the first panel. Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blots were stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for phosphorylated EGFR (Tyr 1173) (Panel 3) and EGFR (C, Panel 4). As a loading control GAPDH was used (Panel 4 in A and B or 5 in C).

Fig 4: Reelin induces Dab1 phosphorylation in NPCs. (A) Western blot of medium conditioned by HEK293T cells transfected with a Reelin-expressing plasmid or empty vector (pcDNA3) and their cell lysates indicates expression and secretion of Reelin. Lysates and conditioned medium (CM) were collected 24 h after transfection. Arrows indicate full-length (FL) Reelin and NR2 and NR6 fragments. (B) WT NPCs treated with 200 μL (+) or 400 μL (++) conditioned medium (CM) from Reelin-transfected HEK293T cells for 10 min have increased Dab1 phosphorylation (pDab1). (C) A total of 20 min treatment with CM from WT NPC cultures increases Dab1 phosphorylation in WT NPCs (n = 4 separate cultures). Pretreatment with Brefeldin A (BFA; 0.75 μg/mL) for 3 h prior to CM treatment reduces Dab1 phosphorylation in WT NPCs, indicating that the state of Dab1 phosphorylation depends on the ability of cells to secrete proteins. Addition of BFA to CM after harvesting does not affect pDab1 induction. Relative band density was normalized to GAPDH. Fold change was 2.19 with p = 0.023 by one-sample t-test.

Fig 5: Presence of ApoER2 diminishes EGF-mediated Dab1 phosphorylation. Western blot analysis of HEK293 (A) cells expressing EGFR and Dab1 (Lanes 1 and 2, ED) or ApoER2, EGFR and Dab1 (Lanes 3 and 4, AED). Cells were either left untreated (Lanes 1 and 3) or treated with human EGF (10 ng/mL) for 3 min (Lanes 2 and 4). Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blots were stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for EGFR phosphorylation (Tyr 1173) (Panel 3) and GAPDH was used as a loading control (Panel 4). (B) The anti-phosphotyrosine signal was normalized to the anti-Dab1 signal and relative levels of phosphorylated Dab1 from 4 independent experiments in HEK293 are presented. The level of Dab1 phosphorylation in the untreated control was set to 1. Data were analyzed by an unpaired, two-tailed t-test, *** p = 0.001; dots, number of experiments. Error bars represent standard deviation.