GRIP1 regulates synaptic plasticity and learning and memory Han L. Tana,1, Shu-Ling Chiua,b,1, Qianwen Zhua,1, and Richard L. Huganira,2 aSolomon H. Snyder Department of Neuroscience and Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bInstitute of Cellular and Organismic Biology, Academia Sinica, 11529 Taipei, Taiwan Contributed by Richard L. Huganir, August 18, 2020 (sent for review July 15, 2020; reviewed by Lin Mei and Peter Penzes) Hebbian plasticity is a key mechanism for higher brain functions, plasticity. For example, synapse-associated protein (SAP97) directly such as learning and memory. This form of synaptic plasticity primarily binds the GluA1 subunit and may promote AMPAR trafficking and involves the regulation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxa- LTP (11–13). Protein interacting with C-kinase 1 (PICK1), a zolepropionic acid receptor (AMPAR) abundance and properties, whereby GluA2 subunit-binding protein, functions to remove AMPAR AMPARs are inserted into synapses during long-term potentiation (LTP) from synapses and causes internalization of synaptic AMPARs, or removed during long-term depression (LTD). The molecular mecha- and deficits in Hebbian plasticity have been reported in PICK1 nisms underlying AMPAR trafficking remain elusive, however. Here we mutant mice (14, 15). show that glutamate receptor interacting protein 1 (GRIP1), an AMPAR- Glutamate receptor interacting protein (GRIP1) is a scaf- binding protein shown to regulate the trafficking and synaptic targeting folding protein that has seven postsynaptic density 95/discs large/ of AMPARs, is required for LTP and learning and memory. GRIP1 is zona occludens (PDZ) domains (16, 17). It interacts directly with Grip1 recruitedintosynapsesduringLTP,anddeletionof in neurons the C terminus of both GluA2 and GluA3 through the fourth blocks synaptic AMPAR accumulation induced by glycine-mediated depo- and fifth PDZ domains. GRIP1 has been shown to regulate the larization. In addition, Grip1 knockout mice exhibit impaired hippocampal surface expression and synaptic stabilization of AMPARs (18, LTP, as well as deficits in learning and memory. Mechanistically, we find 19). Our previous studies, as well as the work of others, have that phosphorylation of serine-880 of the GluA2 AMPAR subunit (GluA2- S880) is decreased while phosphorylation of tyrosine-876 on GluA2 suggested that there might be distinct pools of GRIP1 that dif- (GluA2-Y876) is elevated during chemically induced LTP. This enhances ferentially regulate AMPAR trafficking, although the predomi- the strength of the GRIP1–AMPAR association and, subsequently, the nate role of GRIP1 is to deliver AMPAR to the surface and NEUROSCIENCE – insertion of AMPARs into the postsynaptic membrane. Together, these stabilize them at synapses (20 23). Moreover, this regulation of results demonstrate an essential role of GRIP1 in regulating AMPAR traf- AMPAR trafficking by GRIP1 has been shown to be essential for ficking during synaptic plasticity and learning and memory. certain forms of synaptic plasticity, such as cerebellar LTD and homeostatic scaling (20, 24–26). Nevertheless, the need for synaptic plasticity | LTP | AMPA receptor | GRIP1 | learning and memory GRIP1 in the expression of Hebbian LTP is unknown. Here we investigated the function of GRIP1 in LTP and its he ability of the brain to learn, remember, and adapt requires role in learning and memory. We found that GRIP1 is recruited – Tchanges in synaptic connectivity (1, 2). Synapses are dynamic into synapses with AMPARs, and that the GRIP1 AMPAR in- and subject to cellular mechanisms that strengthen and weaken teraction is enhanced during LTP. Moreover, the loss of GRIP1 these neural connections throughout the lifespan of an organism. blocks the activity-induced accumulation of synaptic AMPARs. Grip1 Associative, or Hebbian, synaptic plasticity is widely thought to Finally, knockout (KO) mice exhibit learning and memory be a key cellular mechanism underlying information storage (3, deficits, likely due to the compromised plasticity at active synapses 4). In Hebbian plasticity, correlated action potential firing be- tween presynaptic and postsynaptic neurons causes long-term Significance potentiation (LTP) of synaptic strength. Conversely, uncorre- lated spiking between presynaptic and postsynaptic neurons in- AMPA receptors (AMPARs) are the principle postsynaptic glu- duces a long-term depression (LTD) at shared synapses (5). The tamate receptors mediating fast excitatory synaptic transmis- molecular mechanisms of Hebbian plasticity are highly complex sion in the brain. Regulation of synaptic AMPAR expression is and currently under intense scrutiny, but the detailed picture required for the expression of synaptic plasticity and normal remains incomplete. brain function. The turnover of AMPARs within synapses is Both LTP and LTD can be mediated by presynaptic mecha- highly dynamic, and the molecular mechanisms underlying nisms, such as enhanced or reduced neurotransmitter release AMPAR trafficking remain unclear. Here we report that GRIP1, probability, as well as by postsynaptic mechanisms, including an AMPAR-binding protein, plays an essential role in delivering changes in the sensitivity, properties, or abundance of postsyn- AMPAR into synapses during synaptic plasticity, particularly in aptic receptors (6, 7). The α-amino-3-hydroxy-5-methyl-4-iso- long-term potentiation. In addition, the deletion of Grip1 cau- xazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) ses synaptic plasticity deficits and impaired learning and are the principle glutamate receptors that mediate the majority of fast memory. Our study reveals a mechanism through which GRIP1 excitatory synaptic transmission in the mammalian central nervous regulates AMPAR trafficking and impacts activity-dependent system, and the postsynaptic abundance of AMPARs is directly synaptic strengthening, as well as learning and memory. proportional to synaptic strength and modulated by a dynamic turnover of these receptors in response to synaptic activity (8, 9). Author contributions: H.L.T., S.-L.C., and R.L.H. designed research; H.L.T., S.-L.C., and Q.Z. Shuttling of AMPARs into and out of the postsynaptic membrane performed research; H.L.T., S.-L.C., and Q.Z. analyzed data; and H.L.T. wrote the paper. are the major mechanisms of NMDA receptor (NMDAR)-depen- Reviewers: L.M., Case Western Reserve University; and P.P., Northwestern University. dent LTP and LTD, respectively, at excitatory synapses (9). AMPARs The authors declare no competing interest. are tetrameric assemblies composed of GluA1-4 subunits, which are Published under the PNAS license. subjected to specialized posttranslational modification and protein in- 1H.L.T., S.-L.C., and Q.Z. contributed equally to this work. teractions that regulate AMPAR conductance and localization (9, 10). 2To whom correspondence may be addressed. Email: [email protected]. Many AMPAR-interacting proteins play critical roles in synaptic First published September 18, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2014827117 PNAS | October 6, 2020 | vol. 117 | no. 40 | 25085–25091 Downloaded by guest on September 27, 2021 in these animals. Taken together, our findings reveal an essential neurons (Fig. 2 A and B). Following cLTP induction, as expec- role of GRIP1 in synaptic plasticity and cognitive functions. ted, in control WT neurons, we observed a significant up- regulation of synaptic AMPARs. However, in Grip1 KO neu- Results rons, glycine treatment failed to induce synaptic enrichment of GRIP1 Is Recruited into Synapses in Response to Chemically Induced AMPARs (Fig. 2 A and C). Therefore, we concluded that GRIP1 LTP. To investigate the role of GRIP1 in regulating AMPAR is essential for AMPAR delivery to synapses during LTP. trafficking during LTP, we first examined GRIP1 expression and subcellular localization before and after LTP induction. Cultured Grip1 KO Mice Exhibit Impaired NMDAR-Dependent LTP. To further rat cortical neurons were treated with glycine to induce chemi- confirm the function of GRIP1 in LTP, we examined the re- cally mediated LTP (cLTP), a well-established stimulation pro- quirement for GRIP1 in LTP by performing intracellular whole- tocol mimicking NMDAR-dependent LTP (NMDAR-LTP) (27), cell electrophysiological recordings. We crossed floxed Grip1 fl/fl and postsynaptic densities (PSDs) were isolated. Consistent with (Grip1 ) mice with the pan-neuronal Nestin-Cre line to delete previous results, we saw significant increases in synaptic GluA1, Grip1 in neurons at embryonic stages (28). GRIP1 protein was fl/fl GluA2, and GluA3 AMPAR subunits after cLTP, while total undetectable in the hippocampus of Nestin-Grip1 mice at AMPAR subunit expression did not change (Fig. 1 A–C). In- postnatal day 21 (Fig. 3A); thus, we performed whole-cell re- triguingly, we observed a synaptic accumulation of GRIP1 protein cordings of LTP in acute hippocampus slices prepared from fl/fl fl/fl following cLTP even though the total GRIP1 level remained un- Nestin-Grip1 mice and control Grip1 littermates at 3 to 4 wk changed (Fig. 1 A–C), suggesting that GRIP1 is recruited into of age. We recorded from CA1 pyramidal neurons because LTP synapses with AMPARs during LTP. at Schaffer collateral-CA1 synapses is well known to be depen- Our previous study suggests that there might be two membrane- dent on NMDARs and expressed primarily by increased synaptic associated pools of GRIP1 with distinct