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. 2017 Oct 3;21(1):84-96.
doi: 10.1016/j.celrep.2017.09.019.

GRIP1 Binds to ApoER2 and EphrinB2 to Induce Activity-Dependent AMPA Receptor Insertion at the Synapse

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Free PMC article

GRIP1 Binds to ApoER2 and EphrinB2 to Induce Activity-Dependent AMPA Receptor Insertion at the Synapse

Sylvia Pfennig et al. Cell Rep. .
Free PMC article

Abstract

Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.

Keywords: AMPA receptors; ApoER2; GRIP1; LTP; ephrinB; synaptic plasticity.

Figures

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Figure 1
Figure 1
ApoER2 and EphrinB2 Are Required for Activity-Induced New AMPA Receptor Insertion (A and B) Potassium chloride (KCl) stimulation co-clusters ephrinBs with ApoER2. Primary hippocampal neurons were isolated from wild-type mice at E17.5 and stimulated with 10 mM KCl for 10 min at 14 DIV to activate neuronal activity. Microscopic images show fluorescent staining of ApoER2 and ephrinB in dendrites. Arrowheads indicate co-clustering of ApoER2 (red) and ephrinB (green) upon KCl stimulation (A). Quantification of ApoER2, ephrinB, and ApoER2-ephrinB colocalized clusters is shown for n = 5 neurons (B). (C and D) Neuronal membrane depolarization by KCl does not alter the expression levels of ApoER2 and ephrinB2. Hippocampal neuron cultures were treated with 10 mM KCl for 10 min at 14 DIV to induce neuronal activity and subjected to subsequent western blot analysis. Western blots showing ApoER2 expression levels and control actin levels (C) and ephrinB2 and control N-cadherin expression levels (D). (E and F) Induction of neuronal activity and activation of ApoER2 induces AMPA receptor insertion in dendritic membranes. Wild-type hippocampal neurons (E17.5) were subjected to AMPA receptor membrane insertion assays. During the assay, neurons were stimulated with 10 mM KCl or concentrated Reelin (Reln) supernatants. Stimulations were conducted for 10 min (KCl) or 3 hr (Reln). Fluorescent images of GluR2 inserted into the dendritic membrane in response to KCl or Reelin (E). Relative fluorescence intensities of newly inserted GluR2 in dendrites of neurons (n = 6) (F). (G and H) ApoER2 is essential for Reelin-induced membrane insertion of AMPA receptors. AMPA receptor membrane insertion was analyzed in wild-type and ApoER2 knockout hippocampal neurons upon stimulation with Reelin for 3 hr. Fluorescent images represent newly inserted GluR2 in dendritic branches of wild-type (+/+) and ApoER2 knockout (ApoER2−/−) neurons upon Reelin stimulation (G). Quantification shows relative intensities of GluR2 insertion in wild-type and ApoER2 knockout neurons (n = 3) (H). (I and J) EphrinB2 mediates the membrane insertion of AMPA receptors upon Reelin stimulation. Control and ephrinB2 knockout neurons were subjected to AMPA receptor membrane insertion assays. Images of dendrites showing newly inserted GluR2 in response to Reelin stimulation in control (Nes-cre−; efnB2lox/lox) and ephrinB2 knockout (Nes-cre+; efnB2lox/lox) hippocampal neurons (I). Quantification of relative GluR2 intensities in neuronal dendrites (n = 3) (J). Scale bars represent 5 μm (A, E, G, and I). Bar graphs show mean ± SEM (shown as error bars). p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S1.
Figure 2
Figure 2
Neuronal Activity Induces Dab1 Phosphorylation at the Synapse through EphrinB2 (A–D) Neuronal activity induces Dab1 phosphorylation in primary hippocampal neurons. Levels of phosphorylated Dab1 were assessed by immunocytochemistry. Fluorescent images showing dendritic pDab1 staining upon stimulation with KCl for 10 min (A) to depolarize the membrane or 25 mM TEA-Cl for 10 min to induce chemical LTP (C). Quantification of relative pDab1 fluorescence intensity is shown in (B; n = 4) and (D; n = 3). (E and F) Hippocampal neurons were stimulated with 10 mM KCl or 25mM TEA-Cl for 10 min and subjected to western blot analysis. Western blot showing increased levels of pDab1 upon KCl stimulation (E). Quantification of relative pDab1 levels (n = 4) (F). (G and H) Induction of neuronal activity causes co-clustering of ephrinB and pDab1 at postsynaptic sites. Hippocampal neurons were treated with KCl and immunocytochemical analysis was performed. Microscopic images show dendritic pDab1 and ephrinB staining. Arrowheads indicate co-clustering of pDab1 (red) and ephrinB (green) upon KCl stimulation (G). Quantification of ephrinB2, pDab1, and ephrinB2-pDab1 colocalized clusters upon KCl stimulation (n = 5 neurons) (H). (I and J) Co-staining of pDab1 (red) and PSD-95 (green) in response to KCl is shown by arrowheads (I). Quantification of PSD-95, pDab1, and colocalized clusters of PSD-95-pDab1 is shown (n = 5 neurons) (J). (K and L) Dab1 phosphorylation upon KCl stimulation depends on ephrinB2. Primary hippocampal neurons were isolated from conditional neuronal specific ephrinB2 knockout embryos at E17.5. At 14 DIV, neurons were stimulated with 10 mM KCl for 10 min and immunocytochemistry for pDab1 was performed. Fluorescent images showing pDab1 in control (Nes-cre−; efnB2lox/lox) and ephrinB2 knockout (Nes-cre+; efnB2lox/lox) neurons stimulated with KCl (K). Quantification of relative pDab1 fluorescence intensity in control and ephrinB2 knockout neurons (n = 3) (L). Scale bars represent 20 μm in (A), (C), and (K) and 5 μm in the higher magnifications in (A), (C), and (K) as well as in (G) and (I). Bar graphs show mean ± SEM (shown as error bars).p < 0.05, ∗∗∗p < 0.001. See also Figures S2–S4.
Figure 3
Figure 3
GRIP1 Binds to ApoER2 and Scaffolds an EphrinB2/ApoER2/AMPA Receptor Complex (A) GRIP1 and ApoER2 co-immunoprecipitate in mouse brain lysates. Total brain lysates from adult wild-type (+/+) and GRIP1 knockout (GRIP−/−) mice were immunoprecipitated with anti-GRIP1, anti-ApoER2, or an unrelated antibody generated in rabbit (Ctrl) and analyzed by western blot for GRIP1 and ApoER2. (B) GRIP1 bridges ApoER2, GluR2, and ephrinB2. Wild-type (+/+) and GRIP1 knockout (GRIP1−/−) brain lysates were immunoprecipitated using anti-ApoER2 antibody, and binding to GluR2 and ephrinB2 was analyzed by western blot. (C) EphrinB ligands mediate the interaction between ApoER2 and GRIP1. Brain lysates from adult control (Nes-cre−; efnB2lox/lox/efnB3+/+) and double ephrinB2/ephrinB3 knockout (Nes-cre+; efnB2lox/lox/efnB3−/−) mice were immunoprecipitated with anti-ApoER2 antibody or normal rabbit immunoglobulin G (IgG; Ctrl) and analyzed by western blot using anti-GRIP1 antibody. (D) Co-immunoprecipitation using anti-ApoER2 antibody or an unrelated rabbit antibody (Ctrl) shows the macromolecular complex formed by ApoER2, GRIP1, GluR2, and ephrinB2. (E and F) Interaction between ApoER2 and GRIP1 is increased upon stimulation with KCl or Reelin in primary hippocampal neuron cultures. Western blots showing GRIP1 co-immunoprecipitation by using an anti-ApoER2 antibody (E). Quantification of relative binding of GRIP1 to ApoER2 upon KCl or Reelin stimulation is shown (n = 3) (F). Bar graphs show mean ± SEM (shown as error bars).∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 4
Figure 4
GRIP1 Is Required for Activity-Induced Dab1 Phosphorylation Mediated by ApoER2 (A and B) Dab1 phosphorylation in response to neuronal activity was examined in primary hippocampal neurons prepared from wild-type (+/+) and GRIP1 knockout (GRIP1−/−) embryos at E15.5. Images of immunocytochemical pDab1 staining in dendrites stimulated with 10mM KCl for 10 min (A). Quantification of pDab1 fluorescence intensities is shown (n = 3) (B). (C) Single-heterozygous neurons for ApoER2 and GRIP1 show normal levels of pDab1. Quantification of relative pDab1 fluorescence intensities in wild-type, ApoER2+/−, and GRIP1+/− neurons (n = 3–6). (D and E) ApoER2 and GRIP1 genetically interact in the phosphorylation of Dab1 upon KCl stimulation. Primary hippocampal neurons from wild-type and ApoER2+/−; GRIP1+/− compound mice were stimulated with KCl, and pDab1 levels were assessed by immunocytochemistry. Fluorescent images represent pDab1 in wild-type (+/+) and ApoER2+/−; GRIP1+/− compound neurons (D). Quantification of relative pDab1 fluorescence intensity in wild-type and ApoER2+/−; GRIP1+/− compound neurons is shown (n = 3) (E). Scale bars represent 20 μm in (A) and (D) and 5 μm in the higher magnifications in (A) and (D). Bar graphs show mean ± SEM (shown as error bars). p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 5
Figure 5
GRIP1 Is Required for ApoER2-Mediated AMPA Receptor Insertion (A and B) GRIP1 is required for AMPA receptor membrane insertion mediated by ApoER2. GluR2 insertion was examined in GRIP1 knockout neurons upon Reelin stimulation. Staining of newly inserted GluR2 in wild-type (+/+) and GRIP1 knockout (GRIP1−/−) dendrites (A). Statistical analysis of GluR2 fluorescence intensities (n = 3) (B). (C) Hippocampal neurons generated from single-heterozygous ApoER2 (ApoER2+/−) and GRIP1 (GRIP+/−) embryos show normal levels of newly inserted GluR2 upon Reelin stimulation (n = 3–4). (D and E) Functional interaction between ApoER2 and GRIP1 is necessary for new AMPA receptor insertion. ApoER2+/−; GRIP1+/− compound hippocampal neurons were subjected to AMPA receptor membrane insertion assays with Reelin stimulation. Microscopic images show newly inserted GluR2 after Reelin stimulation in wild-type (+/+) and ApoER2+/−; GRIP1+/− compound neurons (D). Quantification of relative fluorescence intensities of newly inserted GluR2 in wild-type and compound neurons (n = 3) (E). Scale bars in (A) and (D) represent 5 μm. Bar graphs show mean ± SEM (shown as error bars). p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 6
Figure 6
Ser-9 of EphrinB2 Is Essential for the ApoER2-Mediated Insertion of AMPA Receptors at the Post-synaptic Membrane (A–C) The macromolecular complex consisting of ApoER2, GRIP1, GluR2, and ephrinB2 is disrupted in ephrinB2 S-9 > A mice. Ser-9 of ephrinB2 is necessary for the binding of GRIP1 and GluR2 to ephrinB2. Wild-type (+/+) and ephrinB2 S-9 > A (efnB2 S-9 > A) total brain lysates were immunoprecipitated by using an ephrinB2 antibody or an unrelated goat antibody (Ctrl), and binding of GRIP1 and GluR2 was investigated by western blot (A). The interaction between ApoER2 and GRIP1 is lost in ephrinB2 S-9 > A mutant mice. Wild-type (+/+) and ephrinB2 S-9 > A knockin (efnB2 S-9 > A) brain lysates were immunoprecipitated using anti-ApoER2 antibody or an unrelated rabbit antibody (Ctrl), and the interaction with GRIP1 was analyzed by western blot (B). ApoER2 and GluR2 co-immunoprecipitation in mouse brain lysates is dependent on Ser-9 of ephrinB2. Total brain lysates from adult wild-type (+/+) and ephrinB2 S-9 > A knockin (efnB2 S-9 > A) mice were immunoprecipitated with anti-GluR2 antibody or an unrelated mouse antibody (Ctrl) and analyzed by western blot for ApoER2 and GluR2 (C). (D–G) Ser-9 of ephrinB2 is required for AMPA receptor membrane insertion and functionally interacts with ApoER2 upon stimulation with KCl and Reelin. Wild-type (+/+) and ephrinB2 S-9 > A knockin (efnB2 S-9 > A) neurons were subjected to AMPA receptor membrane insertion assays upon stimulation with 10 mM KCl or concentrated Reelin (Reln) supernatant. Fluorescent images of newly inserted GluR2 staining (D). Fluorescent images of newly inserted GluR2 in wild-type (+/+) and ApoER2+/−; ephrinB2S-9>A/+ compound neurons upon KCl or Reelin (Reln) stimulation (E). Shown are quantification of newly inserted GluR2 intensities in wild-type (+/+) and ephrinB2 S-9 > A (efnB2 S-9 > A) neurons (n = 10) (F) and quantification of fluorescence intensities in wild-type, ApoER2+/−, ephrinB2S-9>A/+, and compound neurons (n = 3–4) (G). Scale bars in (D) and (E) represent 5 μm. Bar graphs show mean ± SEM (shown as error bars). p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S5 and S6.
Figure 7
Figure 7
ApoER2, GRIP1, and EphrinB2 Genetically Interact at the Synapse (A–D) ApoER2, GRIP1, and ephrinB2 genetically interact in LTP. ApoER2+/−; GRIP1+/− compound mice (A and B) and ApoER2+/−; efnB2S-9>A/+ compound mice (C and D) show reduced LTP. LTP was induced by theta burst stimulation (TBS) of Schaffer collaterals and field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region. Representative traces of the compound mice and their respective single-heterozygous and wild-type littermates (+/+) are shown. (ApoER2+/−; GRIP+/− in A; ApoER2+/−; eB2S9>A/+ in C) LTP is significantly reduced in ApoER2+/−; GRIP1+/− compound mice (B) and in ApoER2+/−; efnB2S-9>A/+ compound mice (D) compared to their respective control genotypes at 55–60 min after TBS. (E) GRIP1 regulates ApoER2/ephrinB2 functions at the synapse. Neuronal activity induces ephrinB clustering, leading to the recruitment of Src and its activation, which in turn phosphorylates the adaptor protein Dab1 recruited by ApoER2 receptors. Upon phosphorylation of ephrinB2 in a serine residue (Ser-9), glutamate-receptor-interacting protein 1 (GRIP1) binds to ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors at the synapse in an activity-dependent manner. The formation of such a complex is necessary for activity induced synaptic plasticity. For (A)–(D), bar graphs show mean ± SEM as calculated across slices (shown as error bars). ApoER2-GRIP1: n = 7 wild-type mice (16 slices), 6 ApoER2+/− mice (13 slices), 9 GRIP1+/− mice (16 slices), and 8 ApoER2+/−; GRIP1+/− compound mice (21 slices). ApoER2-ephrinB2: n = 6 wild-type mice (13 slices), 4 ApoER2+/− mice (9 slices), 4 efnB2S-9>A/+ mice (8 slices), and 6 ApoER2+/−; efnB2S-9>A/+ compound mice (15 slices). p < 0.05; ∗∗p < 0.01. See also Figure S7.

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