Reelin induces Erk1/2 signaling in cortical neurons through a non-canonical pathway - PubMed (original) (raw)

Reelin induces Erk1/2 signaling in cortical neurons through a non-canonical pathway

Gum Hwa Lee et al. J Biol Chem. 2014.

Abstract

Reelin is an extracellular protein that controls many aspects of pre- and postnatal brain development and function. The molecular mechanisms that mediate postnatal activities of Reelin are not well understood. Here, we first set out to express and purify the full length Reelin protein and a biologically active central fragment. Second, we investigated in detail the signal transduction mechanisms elicited by these purified Reelin proteins in cortical neurons. Unexpectedly, we discovered that the full-length Reelin moiety, but not the central fragment, is capable of activating Erk1/2 signaling, leading to increased p90RSK phosphorylation and the induction of immediate-early gene expression. Remarkably, Erk1/2 activation is not mediated by the canonical signal transduction pathway, involving ApoER2/VLDLR and Dab1, that mediates other functions of Reelin in early brain development. The activation of Erk1/2 signaling likely contributes to the modulation of neuronal maturation and synaptic plasticity by Reelin in the postnatal and adult brain.

Keywords: Cell Culture; Cell Signaling; Extracellular Signal-regulated Kinase (ERK); Neurodevelopment; Signal Transduction.

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Figures

FIGURE 1.

Purification and analysis of full-length Reelin and its central fragment. A, domain structure of Reelin. Reelin contains an F-spondin homology (FH) region at N terminus, 8 repeats, and a positively charged C terminus. Each repeat contains subdomains A and B, separated by an epidermal growth factor (E)-like motif. Full-length Reelin is cleaved into three fragments. B, Full-length Reelin (FL) and central fragment (CF) proteins secreted in the culture medium of HEK293 GnTI- cells were separated by size exclusion chromatography using Superdex 200 10/300GL. FL Reelin eluted as a single peak with a MW >650 kDa, whereas CF Reelin exhibited two peaks compatible with the formation of a dimer (∼320 kDa) and a tetramer (∼620 kDa).C, Coomassie staining and Western blot analysis of purified proteins separated by SDS-PAGE. Two different batches of FL and CF Reelin were analyzed by Coomassie staining. FL Reelin contained a major band of high MW (∼350–400 kDa), and cleaved fragments of the expected size. Purified CF appeared as a single band of ∼190 kDa, corresponding to the central fragment. Western blot analysis of purified proteins with the N-terminal antibody G10 detects high MW and the N-terminal fragment of FL Reelin. The R5A antibody directed against the central fragment detects high MW and the central fragment of FL Reelin, as well as CF Reelin. D, primary cortical neurons were treated with purified FL and CF Reelin at the indicated concentrations and subjected to the Dab1 phosphorylation assay. Reelin conditioned medium (cR) and mock medium (cM) were used as controls. Both Fl and CF proteins induced robust Dab1 phosphorylation (p-Y).

FIGURE 2.

Activation of Akt and Erk signal transduction pathways by FL Reelin in dissociated cortical neurons. A, 5 DIV mouse cortical neurons were exposed to purified Reelin for 20 min and assayed by Western blotting. Both FL and CF Reelin induced Akt phosphorylation (Ser-473), but only FL Reelin induced Erk1/2 phosphorylation (Thr-202/Tyr-204). B, data were quantified from five independent experiments. The graphs show mean ± S.E. C and D, Time course of Akt and Erk1/2 phosphorylation by FL Reelin and quantification of the results from multiple experiments.

FIGURE 3.

Abnormal Akt and Erk1/2 signaling in juvenile heterozygous_reeler_ and Dab1 KO mice. A, Western blot analysis of forebrain regions from 3–4-week-old wild type (WT) and heterozygous (HT) reeler mice. The levels of phospho-Akt and phospho-Erk1/2 were significantly reduced both the cerebral cortex and hippocampus of reeler mice.B, data were quantified from n_= 9 WT and n = 7 HT mice of the_reeler strain. C, Western blot analysis of cortex and hippocampus from 3–4-week-old WT and HT_Dab1_ KO mice. The phosphorylation levels of Akt and Erk1/2 were decreased significantly in HT mice. D, data were quantified from n = 10 WT,n = 7 HT mice of the Dab1 KO strain.

FIGURE 4.

Activation of Erk1/2 signaling by FL Reelin is independent of the ApoER2/VLDLR-Dab1 canonical pathway. A, Western blot analysis of Reelin-treated 5 DIV cortical neurons in the presence or absence of pharmacological inhibitors. The induction of phospho-Akt by Reelin was not affected by the MEK inhibitor U0126, but was abolished by the PI3K inhibitor LY294002 (30 μ

) and by the SFK inhibitor PP2 (10 μ

). The induction of phospho-Erk1/2 by Reelin was abolished by U0126 (10 μ

) and PP2, but was not affected by LY294002. B, data were quantified from 4–5 independent experiments. C, WT and Dab1_KO cortical neurons were cultured for 5 DIV. FL Reelin induced Akt phosphorylation in WT cortical neurons, but not in Dab1-deficient neurons. Reelin-induced Erk1/2 phosphorylation in both, WT and_Dab1 KO neurons. D, data were quantified from n = 3 WT, _n_= 5 KO independent cultures. E, binding to ApoER2/VLDLR receptors is not required for Reelin-induced Erk1/2 activation. Cortical neurons were treated with FL Reelin in the presence or absence of GST-bound lipoprotein receptor antagonist (RAP) or GST alone as control. Both proteins were added at the 50 μg/ml concentration 15 min prior to Reelin exposure. Akt activation by FL Reelin was completely blocked by RAP, whereas Erk1/2 activation was not affected. F, data were quantified from three independent experiments. G, diagram of the targets of the pharmacological inhibitors used in these experiments. All graphs show mean ± S.E.

FIGURE 5.

Induction of p90RSK phosphorylation by FL Reelin in cortical neurons. A, representative confocal images of cortical neurons treated with control buffer or FL Reelin and processed for immunofluorescence using antibodies against phospho-p90RSK (green) and Map2 (red).B, Reelin significantly increased the percentage of double labeled neurons in the absence of inhibitors and in the presence of LY294002. U0126 pretreatment virtually abolished basal and Reelin-induced levels of phospho-p90RSK. n = 315–345 neurons from 14–15 visual fields per treatment. Scale bars, 50 μm. **, p < 0.01.

FIGURE 6.

Induction of IEGs by FL Reelin. Cortical neurons were treated with FL or CF Reelin for 1 h and mRNA was extracted for quantitative RT-PCR. A, in 4–5 DIV cultures FL Reelin induced Arc mRNA expression in a concentration-dependent manner (10–100 n

), whereas CF Reelin did not. The immunoblot shown below demonstrates induction of Arc protein expression after 3 h of stimulation with higher concentrations of FL, but not CF Reelin. B, FL Reelin induces_Egr_ family of transcription factors, but not_c-Fos_ in 4–5 DIV cortical neurons. CF Reelin has no effect. C, time course of _Arc_and Egr1–3 mRNA expression by FL Reelin 50 n

. D, in 10–12 DIV neurons, FL Reelin induced the expression of all IEGs examined. *,p < 0.05; **, p< 0.01. ***, p < 0.001.

FIGURE 7.

IEGs induction by FL Reelin is mediated by SFKs and MEK. A, quantitative RT-PCR of Reelin-treated cortical neurons in the presence or absence of pharmacological inhibitors.Arc mRNA induction by FL Reelin was completely blocked by U0126 (10 μ

) and PP2 (10 μ

), but not by LY294002 (30 μ

). B, Dab1 is partially required for Reelin-induced Arc mRNA expression. Cortical neurons isolated from newborn _Dab1_KO or WT mice. The induction of Arc mRNA by FL Reelin was reduced in KO compared with WT neurons. C, ApoER2/VLDLR are not required for FL Reelin-induced IEG expression. Cortical neurons were incubated with GST-RAP to block Reelin binding to ApoER2/VLDLR receptors, or to GST alone as control. _Arc_mRNA induction by Reelin was not affected by RAP treatment.D, schematic diagram illustrating signal transduction pathways activated by FL Reelin.

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Reelin induces Erk1/2 signaling in cortical neurons through a non-canonical pathway - PubMed (original) (raw)