ERK activation causes epilepsy by stimulating NMDA receptor activity

Abstract

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1-expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element-binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1-induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Figure 1

Generation and characterization of mice expressing caMEK1 in the brain. (A) Scheme of the βgeo-caMEK1 construct before and after cre recombination. When these single transgenic mice are crossed with Cre transgenic mice, the βgeo cassette is excised and the caMEK1 cDNA is expressed. (B) Southern blot analysis of various tissues of caMEK1^ΔN mice. Ki, kidney; Br, brain; Lu, lung; Te, testes; Sp, spleen. ^*Indicates partially digested, unspecific band. (C) Protein lysates from unrecombined βgeo-caMEK1 and caMEK1^ΔN hippocampi were analysed for MEK1, dually phosphorylated ERK (P-ERK), total ERK and β-actin (loading control) expression. (D) Rotarod performance of 5-week-old βgeo-caMEK1 and caMEK1^ΔN mice (n=6 for each group). Latency time to fall off an accelerating rotarod is indicated±s.e.m. (E) Electrographic seizure recorded from the hippocampus of a caMEK1^ΔN mouse during a behavioural seizure dominated by forelimb myoclonus using in vivo electrophysiological recording with a radiotelemetry system from microwire electrodes positioned in the hippocampus. (F) Quantification of epileptic seizures in caMEK1^ΔN and βgeo-caMEK1 mice. Seven mice per genotype were analysed for 7 h, data are presented±s.e.m.

Figure 2

caMEK1 overexpression does not interfere with neuronal development. Haematoxylin and eosin (H+E) staining (A, B), Luxol–Nissl staining (C, D), immunohistochemistry (IHC) for neurofilament (E, F), neuronal nuclei (NeuN; G, H), Timm's stain (I, J) and IHC for GFAP (K–N), was performed on brain sections from 10-week-old βgeo-caMEK1 and caMEK1^ΔN mice. Rectangles in K and L are magnified in M and N.

Figure 3

Localization of ERK activation in caMEK1^ΔN mice. (A, B) Histological analysis (haematoxylin and eosin staining (H+E)) of hippocampal sections of βgeo-caMEK1 and caMEK1^ΔN mice (8 weeks old). Immunohistochemistry for phosphorylated ERK (P-ERK) on hippocampal (Hc; C, D) and cortical sections (Cx; E, F, F′) of βgeo-caMEK1 and caMEK1^ΔN mice (8 weeks old). Arrowheads in C, D and F indicate P-ERK staining. Double immunofluorescence for MAP-2 (red; G) and P-ERK (green; H) and the merged image (I); and for SNAP-25 (red; J) and P-ERK (green; K) and the merged image (L) on frozen sections encompassing the stratum lucidum of caMEK1^ΔN mice. Scale bar indicates 5 μm. (M) EM photographs of P-ERK staining. ds, dendritic spine; PSD, postsynaptic density; arrow indicate P-ERK staining. Scale bar indicates 200 nm. (N) Schematic representation of EM picture shown in (M).

Figure 4

Increased NR2B protein levels in caMEK1^ΔN mice. (A) RT–PCR analysis of RNA isolated from unrecombined βgeo-caMEK1 and caMEK1^ΔN hippocampi (two independent mice each) for NMDAR2A, NMDAR2B and β-tubulin (loading control) expression. (B) Western analysis of protein lysates from one unrecombined βgeo-caMEK1 and two independent caMEK1^ΔN hippocampi (8 weeks old) for NMDAR1, NMDAR2A, NMDAR2B and β-actin (loading control) expression. (C) Western analysis of protein lysates from unrecombined βgeo-caMEK1 and caMEK1^ΔN hippocampi for eIF4E, phosphorylated eIF4E (P-eIF4E) and β-actin (loading control) expression. (D) Western analysis of protein lysates from SHSY5Y cells (untreated or treated for 1 and 3 h with 10 μM U0126) for eIF4E, phosphorylated eIF4E (P-eIF4E), ERK, phosphorylated ERK (P-ERK) and NR2B expression. ^*Indicates unspecific band. (E) Upper panel: SHSY5Y cells were metabolically labelled with ³⁵S with or without 10 μM U0126. At the indicated time points, NMDAR2B protein was immunoprecipitated (IP), IPs were separated by SDS–PAGE and exposed to X-ray film. Lower panel: Quantification of band intensity using Phosphoimager software. (F) Quantification of epileptic seizures in caMEK1^ΔN mice after a single injection of ifenprodil (1 μg/g body weight). Seizures were scored for 7 h in seven ifenprodil-treated caMEK1^ΔN mice, either before treatment, or on days 1 and 8 after ifenprodil injection. (G) Ifenprodil reduces the paired-pulse index in hippocampal area CA3a of caMEK1^ΔN mutant mice. Traces of extracellular field potential responses as evoked by orthodromic paired-pulse stimulation of area CA3a. Note the smaller population spike amplitude as evoked by the second stimulus in caMEK1^ΔN mutant mice in the presence of 10 μM ifenprodil (right lower trace) as compared to control conditions. (H) Histogram illustrating the significant reduction of the paired-pulse index (PPI, normalized to 100% of control) by ifenprodil in slices from caMEK1^ΔN mutant mice versus control treatment (P<0.01, n=6) and versus ifenprodil treatment in wild type (P<0.05). The difference in the wild type (control treatment versus ifenprodil) is insignificant (P=0.9, n=8). Error bars indicate s.e.m.

Figure 5

EphrinB2 transcription is increased in caMEK1^ΔN hippocampi. (A) Protein lysates from unrecombined βgeo-caMEK1 and caMEK1^ΔN hippocampi (two independent mice) were analysed for CREB, phosphorylated CREB (P-CREB) and β-actin (loading control) protein expression. (B) RT–PCR analysis of RNA isolated from unrecombined βgeo-caMEK1 and caMEK1^ΔN hippocampi (two independent mice each) for ephrinB1 and ephrinB2 expression. (C) Quantitative RT–PCR analysis of RNA isolated from unrecombined βgeo-caMEK1 and caMEK1^ΔN hippocampi for ephrinB2, ephrinB3 and c/EBPbeta expression. (D) Western analysis of protein lysates from unrecombined βgeo-caMEK1 and caMEK1^Δfb hippocampi, or from SHSY5Y cells (untreated or treated for 1 and 3 h with 10 μM U0126) for ephrinB2 and β-actin (loading control) expression. (E) NMDAR2B protein was immunoprecipitated (IP), IPs were separated by SDS–PAGE and western blot analysis for NMDAR2B and phosphorylated tyrosine was performed. Ten-week old mice were used for both mRNA and protein isolation.

Figure 6

EphrinB2 is required for caMEK1-induced NMDAR2B tyrosine phosphorylation and epilepsy. (A) Quantification of epileptic seizures in 8-week old caMEK1^Δfb and caMEK1^Δfb; ephrinB2^Δfb double mutant mice. Five mice per genotype were analysed for 7 h. Error bars indicate s.e.m. (B) Immunohistochemistry for GFAP was performed on brain sections from unrecombined βgeo-caMEK1 and caMEK1^Δfb hippocampi, either wild-type for or lacking EphrinB2. (C) Western analysis of protein lysates from unrecombined βgeo-caMEK1 and caMEK1^Δfb hippocampi, either wild-type for or lacking EphrinB2, for ERK, phosphorylated ERK (P-ERK), ephrinB2, NMDAR2B and β-actin (loading control) expression. Lowest panel: NMDAR2B protein was immunoprecipitated (IP), IPs were separated by SDS–PAGE and western blot analysis for phosphorylated tyrosine was performed. (D) NMDAR2B protein was immunoprecipitated (IP) from protein lysates from unrecombined βgeo-caMEK1 and caMEK1^Δfb hippocampi, either wild-type for or lacking EphrinB2, IPs were separated by SDS–PAGE and western blot analysis for total NMDAR2B protein and NMDAR2B phosphorylated at tyrosine 1472 (P-Y1472) was performed. In A–D, 8–10-week-old mice were used.

Figure 7

Model of regulation of NR2B function by ERK signalling. Schematic representation of the molecular pathways directing transcriptional and translational regulation of NR2B function by MEK1/ERK signalling.