GRIP1 regulates synaptic plasticity and learning and memory

Abstract

Hebbian plasticity is a key mechanism for higher brain functions, such as learning and memory. This form of synaptic plasticity primarily involves the regulation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) abundance and properties, whereby AMPARs are inserted into synapses during long-term potentiation (LTP) or removed during long-term depression (LTD). The molecular mechanisms underlying AMPAR trafficking remain elusive, however. Here we show that glutamate receptor interacting protein 1 (GRIP1), an AMPAR-binding protein shown to regulate the trafficking and synaptic targeting of AMPARs, is required for LTP and learning and memory. GRIP1 is recruited into synapses during LTP, and deletion of Grip1 in neurons blocks synaptic AMPAR accumulation induced by glycine-mediated depolarization. In addition, Grip1 knockout mice exhibit impaired hippocampal LTP, as well as deficits in learning and memory. Mechanistically, we find that phosphorylation of serine-880 of the GluA2 AMPAR subunit (GluA2-S880) is decreased while phosphorylation of tyrosine-876 on GluA2 (GluA2-Y876) is elevated during chemically induced LTP. This enhances the strength of the GRIP1-AMPAR association and, subsequently, the insertion of AMPARs into the postsynaptic membrane. Together, these results demonstrate an essential role of GRIP1 in regulating AMPAR trafficking during synaptic plasticity and learning and memory.

Keywords: AMPA receptor; GRIP1; LTP; learning and memory; synaptic plasticity.

Fig. 1.

GRIP1 is recruited into synapses during cLTP. (A) Representative Western blots of proteins from PSD and total cell lysates (total) isolated from rat cortical neurons treated with (+) or without (−) glycine. (B) Quantification of protein levels in PSD (n = 5; Mann–Whitney U test). (C) Quantification of protein levels in total cell lysates. (n = 8; Student’s t test). (D) Representative Western blots of GRIP1 in P2 and S2 fractions isolated from rat cortical neurons treated with (+) or without (−) glycine. (E) Quantification of GRIP1 protein level in each fraction following cLTP (n = 15 to 16; Mann–Whitney U test). (F) Model of GRIP1 translocation during cLTP. Data are presented as mean ± SEM. n.s., not significant. **P < 0.01.

Fig. 2.

Activity-dependent up-regulation of synaptic AMPARs requires GRIP1 expression. (A) Representative Western blots of proteins from PSD and total cell lysates from WT or Grip1 KO mouse neurons treated with (+) or without (−) glycine. (B) Quantification of protein levels in PSD under basal conditions in WT and Grip1 KO mouse neurons (n = 9 to 11; Student’s t test). (C) Quantification of protein levels in PSD from WT or Grip1 KO mouse neurons treated with (+) or without (−) glycine (n = 15; Mann–Whitney U test). Data are presented as mean ± SEM. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Grip1 KO mice exhibit impaired NMDAR-dependent LTP. (A) Representative Western blots of total cell lysates from hippocampus in Nestin-Grip1^fl/fl mice (Grip1 KO) and control Grip1^fl/fl littermates (WT). (B) Representative evoked EPSCs obtained from CA1 pyramidal neurons before and after LTP induction in response to 0.1-Hz stimulation of Schaffer collaterals. Dash lines represent baseline EPSCs. Solid lines represent EPSCs 40 min after LTP induction. (C) Averaged EPSC amplitudes normalized to baseline responses. Arrow indicates the pairing induction (200 pulses at 2 Hz paired with 0 mV depolarization). (D) Statistics of LTP at 30–50 min (n = 16 cells from 8 control Grip1^fl/fl littermate group; n = 17 cells from 8 Nestin-Grip1^fl/fl mice; Mann–Whitney test). (E) Averaged EPSC amplitudes at baseline and during induction. Data are presented as mean ± SEM *P < 0.05.

Fig. 4.

Grip1 KO mice display impaired learning and memory. (A) Cartoon illustration of the IA task. (B) Quantifications of the latency to cross over to the dark chamber at training and 24 h later in CaMKII-Grip1^fl/fl (Grip1 KO) and control Grip1^fl/fl littermates (WT) (n = 15 WT; n = 13 Grip1 KO; Mann–Whitney U test). (C) Quantification of total, central, and peripheral ambulatory activities in open-field chambers (n = 15 WT; n = 13 Grip1 KO; Mann–Whitney U test). (D) Quantification of the anxiety index, calculated as the activity in the peripheral divided by the activity in the center for each mouse (n = 15 WT; n = 13 Grip1 KO; Student’s t test). Data are presented as mean ± SEM, n.s., not significant; ***P < 0.001.

Fig. 5.

The GRIP1–GluA2 association is enhanced during LTP. (A) Representative Western blots of proteins from P2 in rat cortical neurons treated with (+) or without (−) glycine. (B) Quantification of phospho-Y876 and phospho-S880 levels following cLTP (n = 7 to 12; Student’s t test). (C) GluA2 was immunoprecipitated with specific GluA2 antibody from P2 from rat cortical neurons treated with (+) or without (−) glycine, followed by Western blot analysis of GRIP1 and GluA2. (D) Quantification of relative GRIP1–GluA2 interactions in P2 during cLTP (n = 10; Mann–Whitney U test). (E) Model of GRIP1 regulation of AMPAR trafficking during LTP. Data are presented as mean ± SEM. n.s., not significant; *P < 0.05; ***P < 0.001.