Neuregulin1 (NRG1) signaling through Fyn modulates NMDA receptor phosphorylation: differential synaptic function in NRG1+/- knock-outs compared with wild-type mice

Abstract

We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed that NRG1+/- mutant mice display behavioral abnormalities that are reversed by clozapine, an atypical antipsychotic drug used for the treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the tyrosine kinase receptor for NRG1 in hippocampal neurons, interacts with two nonreceptor tyrosine kinases, Fyn and Pyk2 (proline-rich tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent activation. Furthermore, we show that NRG1 signaling, through activation of Fyn and Pyk2 kinases, stimulates phosphorylation of Y1472 on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypophosphorylated in NRG1+/- mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnormalities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in NRG1+/- mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1 signaling through ErbB4 may contribute to the pathophysiology of schizophrenia through dysfunction of NMDAR modulation. Thus, our data support the glutamate hypothesis of schizophrenia.

Figure 1.

Fyn and Pyk2 interact with ErbB4. Fyn and ErbB4 interact in a yeast two-hybrid growth assay. A, The intracellular domain of ErbB4 physically interacts with a partial cDNA clone comprising the SH2 and SH3 domains of Fyn (ErbB4_i; lane 1) but not with ErbB4 baits containing mutations that inactivate kinase activity (ErbB4_i K751A and ErbB4_i D843A; lanes 2, 3) or with control bait or prey plasmids (lanes 4, 5) (n > 3). B, Full-length ErbB4 (green) and Fyn–V5 (red) colocalize at the cytoplasmic membrane (arrow) when transiently expressed in COS7 cells and visualized by deconvolution microscopy (n > 3) and magnification at 1000×. C, Full-length Fyn–V5 and ErbB4 were stably coexpressed in CHO-K1 cells. Fyn–V5 coimmunoprecipitates with anti-ErbB4 antibodies, as shown by Western blot using anti-V5 antibodies for detection. Stimulation with 7.2 nm NRG1 for 10 min enhanced the interaction (top). ErbB4 is also coimmunoprecipitated with V5 antibody as shown by Western blot using anti-ErbB4 antibodies for detection, and the interaction is enhanced by NRG1 stimulation of the cells as before (middle). No interaction between the two proteins was detected using the control antibody Glu (bottom). D, Pyk2 coimmunoprecipitates with ErbB4 (n = 3) and PSD-95 (n = 2) from mouse brain hippocampus (lanes 1 and 3, respectively) as shown by Western blot with detection using anti-Pyk2. No coimmunoprecipitation was observed with control antibodies (anti-HA; lane 2). IP, Immunoprecipitation.

Figure 2.

NRG1 regulation of the Fyn kinase activity. ErbB4 and Fyn kinase activities were increased in CHO-K1 cells expressing ErbB4 (■) or coexpressing ErbB4 and Fyn–V5 (▴) in response to treatment with 7.2 nm NRG1. A, Fyn kinase activity was measured in the presence of ErbB4 using a substrate with higher affinity for Fyn. Maximum ErbB4 activity was observed within 1 min of stimulation (■), whereas Fyn kinase activity peaked at ∼7 min (▴) in response to NRG1 (n > 3). B, Fyn potentiates NRG1-induced signal transduction activity as measured with an MAPK pathway reporter gene. CHO-K1 cells carrying the pSRE–Luc plasmid and stably expressing either ErbB4 or coexpressing ErbB4 and Fyn–V5 were activated with varying concentrations of NRG1 for 4 h at 37°C and subsequently assayed for luciferase activity. Induction of luciferase activity was increased by approximately twofold (*p < 0.05 after applying the Bonferroni's correction for multiple t tests), whereas the EC₅₀ for NRG1 was unchanged (0.7 vs 1.0 nm, respectively) (n = 3).

Figure 3.

NRG1 activation of ErbB4 regulates Fyn phosphorylation in HEK293 cells coexpressing ErbB4 and Fyn–V5. A, NRG1 stimulation (7.2 nm for 5 min) resulted in phosphorylation of ErbB4 on tyrosine residues and activation of MAPK p44/42 (left top and middle panel, respectively) as shown by Western blot. NRG1 stimulation increased phosphorylation of Fyn on Y420, but had only minor affect on Y531 phosphorylation (right top and middle, respectively) (n = 3). B, NRG1-induced phosphorylation of Fyn Y420 was reduced, but not eliminated, after preincubation of the cells with the Src-family kinase inhibitor PP2 at 25 nm for 16 h (top). The control compound PP3 had no effect. Fyn Y531 phosphorylation was unaffected by NRG1 signaling or the presence of PP2 or PP3 (n = 3). C, NRG1-induced tyrosine phosphorylation of ErbB4 was impaired in the presence of PP2. However, the ErbB4 phosphorylation without NRG1 stimulation was unaffected by PP2 (n = 3).

Figure 4.

NRG1 modulates tyrosine phosphorylation on the NMDAR NR2B subunit in human neuroblastoma cells. Human BE(2)-M17 neuroblastoma cells were differentiated with retinoic acid and stimulated with NRG1α2 (14.4 nm for 10 min). NRG1 stimulation resulted in tyrosine phosphorylation of ErbB4 (second panel), Fyn/Src on Y420 (third panel), Pyk2 on Y402 (fourth panel), and NR2B on Y1472 (bottom panel) as shown by Western blot. Total ErbB4 provided a control for protein loading (top panel) (n > 3).

Figure 5.

NMDAR is hypophosphorylated in NRG1(ΔTM)^+/− and ErbB4^+/− mutant mice, and hypophosphorylation is reversed by clozapine. A, NR2B Y1472 was hypophosphorylated in hippocampal lysates from NRG1(ΔTM)^+/− and ErbB4^+/− mutant mice (top, lanes 2 and 3, respectively) compared with age- and sex-matched wild-type (WT) C57BL/6 mice as shown by Western blot. Fyn/Src Y420 phosphorylation also was reduced in ErbB4^+/− and NRG1(ΔTM)^+/− mutant mice (bottom, lanes 2 and 3, respectively). Hippocampal lysates from Fyn^−/− mice served as a control for NR2B Y1472 hypophosphorylation, as well as for Fyn/Src Y420 phosphorylation (fourth lane, top and bottom panels, respectively). Data are representative of five to six animals in at least three independent experiments; 25 μg of total protein was loaded per lane. B, Clozapine reverses NR2B hypophosphorylation in NRG1(ΔTM)^+/− mice. NR2B Y1472 phosphorylation (top) was increased 2.5- to 3-fold in NRG1(ΔTM)^+/− mice when normalized against total loading of NR2B (bottom). The same dose of clozapine had no effect on NR2B Y1472 phosphorylation (top) when administered to age- and sex-matched wild-type mice. Data representative of two to three animals in at least two independent experiments; 20 μg of total protein was loaded per lane.

Figure 6.

Effects of NRG-1 gene deletion on basal synaptic transmission. A, PPF was measured by applying two closely spaced stimuli, and the ratio of the second response to the first response was calculated. PPF was enhanced in NRG1(ΔEGF)^+/− slices (open circles; n = 67) compared with wild-type slices (filled squares; n = 76). B, PPF was measured in untreated NRG1(ΔEGF)^+/− slices (solid line; n = 18), then slices were treated for 30 min with 1 nm NRG1β1, and facilitation was measured again (dotted line). C, The rate of synaptic fatigue during a 100 Hz stimulus train was fit by a single-exponential function. The resulting rates were similar between wild-type (filled squares; 5.5 stimuli; n = 24) and NRG1^+/− (open circles; 6.2 stimuli; n = 17) slices.

Figure 7.

Effects of NRG1 gene deletion on LTP. A, LTP was induced by five trains of 100 Hz stimuli lasting 200 ms, with each train separated by 10 s. Data are normalized to baseline values preceding LTP induction, and mean potentiation was measured 45 min after tetanus. LTP was not significantly different between wild-type (filled squares; n = 24) and NRG1(ΔEGF)^+/− (open circles; n = 17) slices. B, LTP was induced by theta-burst stimulation (TBS) consisting of 10 sets of four stimuli given at 100 Hz, with each set separated by 200 ms. Theta-burst-induced LTP was significantly diminished in NRG1(ΔEGF)^+/− slices (open circles; n = 13) compared with wild-type slices (filled squares; n = 13).

Figure 8.

Effects of exogenously applied NRG1 on LTP in wild-type and NRG1(ΔEGF)^+/− slices. A, NRG1β1 was applied to wild-type slices at varying concentrations for 30 min after a stable baseline of at least 10 min was achieved. LTP was induced as described previously by a tetanic train. NRG1β1 inhibited tetanus-induced LTP in wild-type slices, ∼50% at 0.1 nm (n = 18), and completely ablated it at 1 nm (n = 8). B, NRG1β1 enhanced tetanus-induced LTP at low concentrations (0.1 nm; n = 9) in NRG1(ΔEGF)^+/− slices but inhibits LTP at higher concentrations of 1 nm (n = 8) and 10 nm (n = 5). C, NRG1β1 inhibited theta-burst-induced LTP in wild-type slices, ∼50% at 0.1 nm (n = 4) and completely at 1 nm (n = 4). D, Acute treatment with NRG1β1 dose-dependently reversed deficits in theta-burst-induced LTP in NRG1(ΔEGF)^+/− slices. The deficit was partially ameliorated with 0.1 nm (n = 11) and 10 nm (n = 5) NRG1β1 and was maximally reversed with 1 nm NRG1β1 (n = 15).