GRIP1 regulates and learning and

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 functions, plasticity. For example, -associated (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 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 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 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 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 functions: the intracellular AMPAR abundance (6). Furthermore, LTP and AMPAR traf- GRIP1 retains AMPARs intracellularly, while the plasma membrane- ficking in CA1 neurons have been functionally linked to animal associated GRIP1 anchors AMPARs at the cell surface (20). In learning and memory (29, 30). Our data reveal an ∼50% de- addition, there is abundant non–membrane-associated GRIP1 in crease in LTP expression in CA1 pyramidal neurons of Nestin- fl/fl fl/fl the cytosol. To examine how these pools change during LTP, we Grip1 compared with control Grip1 littermates (Fig. 3 B–D). performed subcellular fractionation to determine GRIP1 sub- This reduced potentiation lasted at least 50 min in neurons of fl/fl + cellular distribution on cLTP. Our data show that cLTP did not Grip1 ; Nestin-Cre mice compared with control neurons from fl/fl significantly change GRIP1 levels in either cytosol (S2) or mem- Grip1 ; Nestin-Cre-mice (Fig. 3D). The impaired LTP in Nestin- fl/fl brane (P2) fractions (Fig. 1 D and E). Together, these results in- Grip1 mice is not caused by inefficient induction, because dicate that synaptic enrichment of GRIP1 after cLTP is due to excitatory postsynaptic current (EPSC) amplitudes at baseline fl/fl translocation from the intracellular membrane pool, likely from and during LTP induction were comparable in Nestin-Grip1 fl/fl the endosomes, rather than from the cytosolic non–membrane- mice and control Grip1 littermates under our induction pro- associated pool (Fig. 1F). tocol (Fig. 3E). Together, these data show that GRIP1 is re- quired for induction and maintenance of LTP. Activity-Dependent Up-Regulation of Synaptic AMPARs Requires GRIP1 Expression. To directly determine the functional roles of Grip1 KO Mice Display Impaired Learning and Memory. Synaptic GRIP1 in LTP, we next used neurons derived from GRIP1 plasticity is a key molecular mechanism underlying learning and conditional KO mice. As has been shown previously (20, 25), memory. In light of the impaired synaptic plasticity observed in GRIP1 protein was completely ablated in neurons transduced Grip1 KO neurons, we next evaluated the cognitive functions of with lentiviruses expressing Cre recombinase (EGFP-IRES-Cre) Grip1 KO mice. As Nestin-Cre mice exhibit cognitive behavioral (Fig. 2A). In unstimulated basal conditions, synaptic GluA1, abnormalities, including reduced contextual and cued conditioned GluA2, and GluA3 expression in Grip1-deleted neurons was fear responses (31, 32), we used calmodulin-dependent kinase II fl/fl comparable to that in control EGFP-expressing wild-type (WT) (CaMKII)-Grip1 mice to perform behavior experiments, with

AB C PSD Total 250 150 n.s. Glycine - + - + 200 ** GluA1 ** 100 150 ** ** GluA2 100 50 GluA3 (% Control) 50 (% Control) PSD enrichment GRIP1 0 enrichment Total 0

Tubulin Fig. 1. GRIP1 is recruited into synapses during cLTP.

GluA1 GluA2 GluA3 GRIP1 GluA1 GluA2 GluA3 GRIP1 (A) Representative Western blots of proteins from PSD and total cell lysates (total) isolated from rat DE F LTP cortical neurons treated with (+) or without (−) gly- 150 n.s. Control cine. (B) Quantification of protein levels in PSD (n = Glycine - + 5; Mann–Whitney U test). (C) Quantification of pro- GRIP1 100 tein levels in total cell lysates. (n = 8; Student’s t test). (P2) (D) Representative Western blots of GRIP1 in P2 and GRIP1 50 S2 fractions isolated from rat cortical neurons treat- ed with (+) or without (−) glycine. (E) Quantification (S2) (% Control) of GRIP1 protein level in each fraction following cLTP 0 Total enrichment Total = – P2 S2 (n 15 to 16; Mann Whitney U test). (F) Model of GRIP1 (P2) (S2) AMPAR GRIP1 translocation during cLTP. Data are presented GRIP1 as mean ± SEM. n.s., not significant. **P < 0.01.

25086 | www.pnas.org/cgi/doi/10.1073/pnas.2014827117 Tan et al. Downloaded by guest on September 27, 2021 Grip1 AB KO (Conditional) EGFP Cre-EGFP 150 Glycine - + - + n.s. Total GRIP1 100 GluA1

50 GluA2 (% WT)

PSD PSD enrichment GluA3 0 GluA1 GluA2 GluA3 GluN1 Grip1 KO C 150 GluA1 GluA2 GluA3 *** ** *

100 Fig. 2. Activity-dependent up-regulation of synaptic AMPARs requires GRIP1 expression. (A) Representative Western blots of proteins from PSD and total cell ly- sates from WT or Grip1 KO mouse neurons treated 50 (% Control) with (+) or without (−)glycine.(B) Quantification of

protein levels in PSD under basal conditions in WT and NEUROSCIENCE PSD enrichment Grip1 KO mouse neurons (n = 9 to 11; Student’s t test). (C) Quantification of protein levels in PSD from WT or 0 Grip1 KO mouse neurons treated with (+) or without Glycine - + - +e - +e - +e - +e - +e (−)glycine(n = 15; Mann–Whitney U test). Data are Grip1 Grip1 Grip1 presented as mean ± SEM. n.s., not significant. *P < WT KO WT KO WT KO 0.05; **P < 0.01; ***P < 0.001.

fl/fl Grip1 littermates serving as controls. We chose to use an in- study showed that regulation of GluA2 Y876 phosphorylation hibitory avoidance (IA) task, as IA learning induces LTP and could gate GluA2 S880 phosphorylation (40). During cerebellar synaptic AMPAR incorporation in the hippocampus and thus is LTD, dephosphorylation of GluA2 Y876 occurs, which in- dependent on hippocampal function (30, 33–35). As described creases GluA2 phospho-880, decreases GRIP1 interactions, and previously (36), adult mice were habituated to a chamber divided thus accelerates GluA2 internalization (40). We recently showed into two separate compartments: light and dark. When placed in fl/fl fl/fl that phosphorylation of GluA2 Y876 directly increases GRIP1 the light side, both CaMKII-Grip1 mice and control Grip1 binding to GluA2, and that this regulation is necessary for syn- A B littermates entered the dark side after a short time (Fig. 4 and ). aptic upscaling (41). At 24 h after training, during which a mild foot shock was delivered To gain insight into the mechanisms of these actions, we first after the mice entered the dark side, control mice showed a sig- examined the phosphorylation levels of GluA2 S880 and Y876 nificant longer step-through latency, indicating a clear IA memory during cLTP. Following glycine treatment, we observed a signifi- A B CaMKII-Grip1fl/fl (Fig. 4 and ). However, mice failed to learn cant increase in GluA2 phospho-Y876 level while phospho-S880 the IA task and showed no significant difference between before A B A B level was decreased (Fig. 5 and ). Given that phosphorylation and after the IA training (Fig. 4 and ). of GluA2 Y876 increases GRIP1 binding but phospho-S880 in- To rule out potential behavioral interference, such as general hibits it, these results imply that the GRIP1–GluA2 association motor activity and anxiety, we also performed open-field test fl/fl fl/fl might be enhanced during LTP. To confirm this, we performed with the CaMKII-Grip1 mice and their control Grip1 lit- coimmunoprecipitation experiments to directly examine GRIP1- termates. No significant differences in locomotion and time spent in the center vs. the periphery of the chamber were ob- GluA2 binding. Since GluA2 is a transmembrane protein, we used served in these mice (Fig. 4 C and D), supporting our conclusion the membrane fraction (P2) to avoid the artificial binding of cy- that GRIP1 is specific and essential for hippocampal-dependent toplasmic GRIP1 with GluA2. As expected, we observed increased learning and memory. coimmunoprecipitation between GRIP1 and GluA2 following cLTP treatment (Fig. 5 C and D), indicating a stronger GRIP1– GRIP1-GluA2 Association Is Enhanced during LTP. Finally, we exam- GluA2 association during LTP. Taken together, these results show ined the underlying molecular mechanism through which GRIP1 that GRIP1–GluA2 interaction is enhanced during LTP, involving regulates AMPAR trafficking during LTP. GRIP1 directly binds coordination of GluA2-Y876 and GluA2-S880 phosphorylation. with the C-termini of GluA2/3 AMPAR subunits, and the in- teraction is highly regulated by activity and plays critical roles in Discussion AMPAR trafficking (10). For example, during cerebellar LTD, In the present study, we have demonstrated an essential role of GluA2 S880 is phosphorylated, which disrupts GRIP1 binding GRIP1 in synaptic plasticity and learning and memory. We found to GluA2 and in turn increases GluA2–PICK1 interaction to that GRIP1 traffics with AMPARs from intracellular membranes promote AMPAR endocytosis (37–39). In addition, another into synapses during LTP, and that the interaction between

Tan et al. PNAS | October 6, 2020 | vol. 117 | no. 40 | 25087 Downloaded by guest on September 27, 2021 AB Nestin-Cre - +

GRIP1 20 pA 10 ms GluA2/3 WT Grip1 KO CD WT 2.5 * 2.5 Grip1 KO 2.0

2.0 1.5

1.5 1.0 16/8 at 30-50 min 0.5 1.0 17/8

Norm. EPSC amplitude 0.0 Norm. EPSC amplitude Norm. EPSC amplitude Fig. 3. Grip1 KO mice exhibit impaired NMDAR- 0 10 20 30 40 50 WT Grip1 KO dependent LTP. (A) Representative Western blots of E Time (min) total cell lysates from hippocampus in Nestin- 80 Grip1fl/fl mice (Grip1 KO) and control Grip1fl/fl litter- Baseline (0.1Hz) mates (WT). (B) Representative evoked EPSCs obtained from CA1 pyramidal neurons before and 60 WT Grip1 after LTP induction in response to 0.1-Hz stimulation KO of Schaffer collaterals. Dash lines represent baseline 40 2 Hz at 0 mV EPSCs. Solid lines represent EPSCs 40 min after LTP induction. (C) Averaged EPSC amplitudes normalized to baseline responses. Arrow indicates the pairing 20 induction (200 pulses at 2 Hz paired with 0 mV de- polarization). (D) Statistics of LTP at 30–50 min (n = fl/fl

EPSC amplitude (pA) 16 cells from 8 control Grip1 littermate group; n = fl/fl 0 17 cells from 8 Nestin-Grip1 mice; Mann–Whitney test). (E) Averaged EPSC amplitudes at baseline and 0 50 100 150 200 during induction. Data are presented as mean ± SEM Stimulus number *P < 0.05.

GRIP1 and AMPAR is also strengthened as a consequence of with the findings of previous studies, indicate that there are distinct increased GluA2-Y876 phosphorylation and decreased GluA2- but overlapping characteristic features of Hebbian and homeostatic S880 phosphorylation. Grip1 KO neurons have impaired LTP, synaptic plasticity (45, 46). and Grip1 KO mice exhibit learning and memory deficits. GRIP1 binds directly with GluA2 and GluA3 AMPAR sub- The regulation of GRIP1 in AMPAR trafficking has been a units but not with the GluA1 subunit; however, the role of controversial subject. Some studies have shown that GRIP1 de- GRIP1 in activity-dependent AMPAR trafficking is not subunit- livers AMPARs to the cell surface and stabilizes AMPARs at specific. The cLTP-induced increases in synaptic AMPAR levels, synapses (18, 42), while other studies have suggested that GRIP1 including levels of GluA1, GluA2 and GluA3 subunits, are retains AMPAR intracellularly (43, 44). We recently provided blocked in Grip1 KO neurons. Synaptic incorporation of calcium- evidence suggesting the presence of two membrane-associated permeable AMPARs (CP-AMPARs) following LTP induction, pools of GRIP1, one pool associated with the plasma mem- mostly with GluA1 homomers, has been reported (47, 48); how- brane to anchor AMPARs on the cell surface and the other pool ever, these findings have not been consistently replicated (49, 50). in the cytoplasm to retain AMPARs within intracellular compart- Furthermore, the presence of CP-AMPARs at synapses during ments (20). These two distinct pools function cooperatively to reg- LTP, if any, is very brief before their replacement by GluA2- ulate AMPAR trafficking. For example, in homeostatic up-scaling, containing AMPARs, which are thought to be essential for LTP in which surface AMPARs are greatly up-regulated, synaptic GRIP1 maintenance (48, 51). Therefore, GluA2-containing AMPARs is increased and the association between GRIP1 and synaptic play a major role in LTP, and GRIP1 regulates GluA1 expression AMPARs is strengthened, while intracellular GRIP1–AMPAR by controlling GluA1-GluA2 heteromers. Notably, LTP is partially interaction is reduced. Furthermore, the increase in membrane- impaired in hippocampus slices of Grip1 KO mice. Some com- associated GRIP1 is accompanied by a decrease in cytosolic pensatory involvement of other AMPAR-binding proteins, such as non–membrane-associated GRIP1, indicating a translocation of the transmembrane AMPAR regulatory proteins, GRIP2, a GRIP1 GRIP1 from the cytosol to membrane. Intriguingly, we did not homolog, PICK1, or GluA1-interacting partners such as SAP-97 or observe any changes in cytosolic GRIP1 during LTP despite an protein 4.1 N, also may contribute to this phenotype (10, 52). increase in synaptic GRIP1 level, suggesting that the synaptic AMPARs are subject to posttranslational modifications, in- accumulation of GRIP1 during LTP is caused by translocation of cluding phosphorylation, ubiquitination, and palmitoylation. These GRIP1 from intracellular membrane compartments, likely from modifications have a significant impact on AMPAR trafficking, endosomes, rather than from the cytosolic non–membrane-associated primarily by affecting the binding of other proteins with AMPARs. pool (Fig. 5E). Although GRIP1 is required for both LTP and Phosphorylation is the most extensively studied modification, and tetrodotoxin (TTX)-induced up-scaling, the mechanisms through numerous phosphorylation sites have been characterized (9). For which GRIP1 regulates these processes differ. These data, together example, GluA1-S567 and GluA1-S831 can be phosphorylated by

25088 | www.pnas.org/cgi/doi/10.1073/pnas.2014827117 Tan et al. Downloaded by guest on September 27, 2021 AB *** 100

80

60 n.s.

40 0.7 mA 2 sec 20

0 Step through latency (sec) latency through Step

Test Test Training Training CDWT Grip1 KO n.s. WT 5000 Grip1 KO 0.20 WT n.s. n.s. Grip1 KO Fig. 4. Grip1 KO mice display impaired learning and 4000 memory. (A) Cartoon illustration of the IA task. (B) 0.15 Quantifications of the latency to cross over to the dark chamber at training and 24 h later in CaMKII- 3000 Grip1fl/fl (Grip1 KO) and control Grip1fl/fl littermates = = – 0.10 (WT) (n 15 WT; n 13 Grip1 KO; Mann Whitney U test). (C) Quantification of total, central, and pe- 2000 ripheral ambulatory activities in open-field chambers

Anxiety index (n = 15 WT; n = 13 Grip1 KO; Mann–Whitney U test). 0.05 Locomotor activity 1000 n.s. (D) Quantification of the anxiety index, calculated as the activity in the peripheral divided by the activity in

(Peripheral / Center activity) the center for each mouse (n = 15 WT; n = 13 Grip1 0 0.00 ’ ± NEUROSCIENCE (# of beam breaks over 30 mins) KO; Student s t test). Data are presented as mean Total Center Peripheral SEM, n.s., not significant; ***P < 0.001.

Ca2+/CaMKII (53, 54), and protein kinase C (PKC) phosphory- the synapses and LTP (36). Together, our findings elucidate the lates GluA2-S863 and GluA2-S880 (37, 55). Along with serine/ function of GRIP1 in synaptic plasticity and may shed light on the threonine phosphorylation, tyrosine phosphorylation of AMPARs mechanisms underlying neurodevelopmental disorders character- also has been reported. GluA2-Y876 and GluA3-Y881 can be ized by synaptic and cognitive abnormalities. phosphorylated by the Src family tyrosine kinases (41, 56). These various phosphorylation modifications have distinct but significant Materials and Methods roles in modulating the properties, function, and trafficking of Neuronal Culture. Cortical neurons from embryonic day 18 rat or mouse pups AMPARs (9, 10). The binding affinity of GRIP1 for GluA2 is were plated on poly-L-lysine–coated tissue culture dishes at a density of 2 largely affected by the phosphorylation of GluA2-S880 and GluA2- 75,000 cells/cm in 5% horse serum (Invitrogen) containing Neurobasal me- Y876. GluA2-S880 phosphorylation prevents GRIP1 binding, while dium (Invitrogen) supplemented with 2% B-27, 2 mM Glutamax, and 50 U/mL – pen-strep. Neurons were switched to 1% horse serum containing Neurobasal GluA2-Y876 phosphorylation enhances GRIP1 GluA2 interaction medium after neurons grew attached to the plate, and were then treated with (40, 41). During cLTP, we observed a concomitant decrease in FDU (5 mM 5-fluoro-2′-deoxyuridine and 5 mM uridine) to inhibit glia prolif- GluA2-S880 phosphorylation and an increase in phosphorylated eration at day in vitro (DIV) 5, and fed twice per week with glia-conditioned GluA2-Y876. In addition, we found a stronger association of 1% horse serum containing Neurobasal medium with supplements. GRIP1 with GluA2 during LTP. These data support a model in which more GRIP1 binds with AMPARs as a result of coordinated Glycine-Induced LTP. Cortical neurons at DIV 18 to 20 were first incubated in GluA2-S880 and GluA2-Y876 phosphorylation to deliver them into artificial cerebrospinal fluid (ACSF) containing 143 mM NaCl, 5 mM KCl, synapses during LTP (Fig. 5E). Interestingly, a single point mutation 10 mM Hepes, 10 mM glucose, and 2 mM CaCl2 (pH 7.4) supplemented with 1 mM MgCl2, 500 nM TTX, 20 μm bicuculine, and 1 μm strychnine at 37 °C for that prevents phosphorylation of Y876 alone is insufficient to μ block LTP (41). It is possible that these two sites may function 30 min, and then treated with 200 m glycine in ACSF with the same sup- plements except MgCl for 10 min, followed by a 20-min recovery in ACSF cooperatively and redundantly. 2 supplemented with 1 mM MgCl2, 500 nM TTX, 20 μm bicuculine, and 1 μm Because GRIP1 is critical for synaptic plasticity, and synaptic strychnine. The cells were then harvested for further experiments. plasticity is crucial for cognitive function, we examined the role of GRIP1 in learning and memory with the knowledge that Lentivirus Generation. The lentiviruses were generated based on a protocol GRIP1 is essential for LTP. We found that Grip1 KO mice have provided by Carlos Lois of MIT, Cambridge, MA (57). Targeted cDNA for viral significant impairments in learning and memory, supporting the expression was first cloned into a pFUW vector containing a ubiquitin pro- notion that regulation of AMPAR trafficking during synaptic moter and then cotransfected with Δ8.9 and VSVG packaging constructs into plasticity is important for brain function (1). Indeed, various HEK293T cells when the cells were 90% confluent. At 1 d after transfection, the supernatant of HEK 293T cells containing released viruses was collected GRIP1 single nucleotide polymorphisms have been reported to × strongly associate with autism (19), and these variants have altered and concentrated by centrifugation at 250,000 gfor2hat4°C.Thesuper- natant was discarded, and the pellet was resuspended in Neurobasal medium. interactions with GluA2/GluA3 and thus affect AMPAR traf- ficking. In addition, a recent study on the GRIP1-binding protein Subcellular Fractionation. Rat or mouse cortical neurons were harvested in GRASP1 showed that intellectually disability-associated GRASP1 homogenate buffer (320 mM sucrose, 5 mM sodium pyrophosphate, 1 mM mutations have convergent impairments in their interactions with EDTA, 10 mM Hepes pH 7.4, 200 nM okadaic acid, 2.5 mM sodium orthovanadate, GRIP1, which in turn blocks endosomal delivery of AMPARs to and protease inhibitor mixture [Roche]) and homogenized using a 26-gauge needle.

Tan et al. PNAS | October 6, 2020 | vol. 117 | no. 40 | 25089 Downloaded by guest on September 27, 2021 P2 anti-GluA3 pAb (JH4300, made in-house), anti-GRIP1 mAb (BD Biosciences), AB200 anti-GRIP1 pAb (Chemicon), and anti-GRIP1 pAb (JH2260, made in-house). Glycine - + * 150 fl/fl fl/fl pY876 Electrophysiology. Paired littermates of Nestin-Grip1 and Grip1 mice 100 (both males and females) at postnatal day 19 to 28 were anesthetized with pS880 *** the inhalation of isoflurane before decapitation. Then 300-μm-thick transverse 50 hippocampal slices were prepared with a vibratome (Leica VT 1200s) in ice-cold (% Control)

GluA2/3 enrichment Total oxygenated (95% O2/5% CO2) dissection buffer containing 210 mM sucrose, 7 mM 0 glucose, 26.2 mM NaHCO , 2.5 mM KCl, 1 mM NaH PO ,and7mMMgSO. Slices pY876 pS880 3 2 4 4 were recovered in a submersion chamber filled with oxygenated ACSF (119 mM CD250 NaCl, 26.2 mM NaHCO3, 11 mM glucose, 2.5 mM KCl, 1 mM NaH2PO4,2.5mM Input IgG IP:GluA2 * 200 CaCl2,and1.3mMMgSO4) at 36 °C for 30 min before recording. Glycine - + - + μ 150 For LTP recordings, slices were perfused in ACSF in the presence of 100 M picrotoxin at room temperature. Hippocampal CA1 neurons were patched by GRIP1 100 glasspipettes(4to5MΩ) which were filled with internal solution (115 mM Cs- GluA2 (% Control) 50 MeSO3,0.4mMEGTA,5mMTEA-Cl,2.8mMNaCl,20mMHepes,3mMMg-ATP, 0.5 mM Na -GTP, 10 mM Na phosphocreatine, and 5 mM QX-314, pH 7.2; os- 0 2 GRIP1-GluA2 binding − Glycine - + molality 295 to 300 mOsm). Cells were held at 70 mV, and responses were evoked at 0.1 Hz by electrical stimulation (0.1 ms, 8 to 20 μA) via a bipolar E electrode positioned at the midline of the Schaffer collateral. LTP was induced by a train of 200 pulses administered at 2 Hz paired with 0 mV depolarization. Signals were measured with MultiClamp 700B amplifier and digitized using a Digidata 1440A digitizer (Molecular Devices). Data acquisition were performed LTP with pClamp 10.5 software and digitized at 10 kHz. Data are presented as re- Control sponses averaged at 1-min intervals and then normalized to the average of

GRIP1 baseline response. Access resistance (Ra) was monitored throughout the re- cording. Cells in which the R > 20 MΩ or the R varied by >20% were discarded. AMPAR a a

pS880 Behavior Assays. Adult male and female CaMKII-Grip1fl/fl mice and Grip1fl/fl pY876 littermates (4 to 5 mo old) were grouped and housed with both genotypes. For the open-field test, mice were placed in a photobeam- equipped plastic – Fig. 5. The GRIP1 GluA2 association is enhanced during LTP. (A) Repre- chamber (45 × 45 cm, PAS open-field system; San Diego Instruments) for free sentative Western blots of proteins from P2 in rat cortical neurons treated exploration for 30 min. The peripheral area (425 cm2) was defined by the two − with (+) or without ( ) glycine. (B) Quantification of phospho-Y876 and side-photobeams, 1-2 and 15-16, while the central area (1,600 cm2) was defined = ’ phospho-S880 levels following cLTP (n 7 to 12; Student s t test). (C) GluA2 by photobeams 3 to 14 at each direction. Movements and rearing behavior were was immunoprecipitated with specific GluA2 antibody from P2 from rat tracked using the SDI Photobeam Activity System (San Diego Instruments). − cortical neurons treated with (+) or without ( ) glycine, followed by Western For the IA task, mice were handled for 3 min each day for 4 consecutive days – blot analysis of GRIP1 and GluA2. (D) Quantification of relative GRIP1 GluA2 before testing. The step-through IA apparatus (Gemini Avoidance System) = – interactions in P2 during cLTP (n 10; Mann Whitney U test). (E) Model of consists of a rectangular chamber divided into two separate compartments GRIP1 regulation of AMPAR trafficking during LTP. Data are presented as (light and dark) connected by a guillotine-style door. The latency to crossover ± < < mean SEM. n.s., not significant; *P 0.05; ***P 0.001. was recorded automatically. On day 1 (habituation), an individual mouse was placed in the light compartment for free exploration until it entered into the dark side and the door immediately closed. The mouse was promptly put back The homogenate was then centrifuged at 800 × gfor10minat4°CtoyieldP1and to the home cage after entering the dark side. On day 2 (training), the mouse S1. S1 was centrifuged at 17,000 × gfor20mintoyieldP2andS2.P2wasthen was reintroduced to the light compartment, and a scrambled 0.7-mA, 2-s foot resuspended in water adjusted to 4 mM Hepes pH 7.4, followed by 30 min of shock was delivered immediately after the mouse crossed to the dark com- agitation at 4 °C. Suspended P2 was centrifuged at 25,000 × g for 20 min at partment. The mouse was then put back into the home cage. On day 3 (test), 4 °C. The resulted pellet was resuspended in 50 mM Hepes pH 7.4, mixed with at 24 h after the training, the mouse was reintroduced into the light com- an equal volume of 1% Triton X-100, and agitated at 4 °C for 10 min. The PSD partment, and the latency to step through to the dark side was recorded as a fraction was generated by centrifugation at 32, 000 × g for 20 min at 4 °C. measure of memory retention.

Coimmunoprecipitation. The P2 membrane fraction was lysed in PBS con- Statistical Analysis. All statistical analyses were performed in GraphPad Prism – taining 50 mM NaF, 5 mM sodium pyrophosphate, 1% Nonidet P-40, % 7. The Shapiro Wilk test was first performed to determine whether the data sodium deoxycholate, 1 μM okadaic acid, 2.5 mM sodium orthovanadate, were normally distributed, and then comparisons were made using para- and protease inhibitor mixture (Roche). The anti-GluA2 antibody (032.19.9, metric or nonparametric tests, as appropriate. For data that passed the normality test, statistical significance was determined by unpaired two- made in- house) or control IgG antibody was precoupled to protein A Sepharose tailed Student’s t test as indicated in the figure legends. For data that did beads and incubated with 200 μg P2 protein in lysis buffer at 4 °C for 2 h. The not pass the normality test, statistical significance was determined by the beads were then washed in lysis buffer six times, followed by 2× sodium unpaired two-tailed Mann–Whitney test as indicated in the figure legends. dodecyl sulfate (SDS) loading buffer elution. Bound proteins were resolved by All data are presented as mean ± SEM. SDS-polyacrylamide gel electrophoresis for Western blot analysis. Data Availability. All study data are included in the paper. Antibodies. The following antibodies were used: anti–beta-tubulin mAb (Sigma-Aldrich), anti-GluA1 N-terminal antibody mAb (4.9D, made in-house), ACKNOWLEDGMENTS. We thank all members of the R.L.H. laboratory for anti-GluA2 N-terminal antibody mAb (032.19.9, made in-house), anti-GluA2 discussions and support, particularly Drs. Kacey E. Rajkovich and Adeline J. H. phospho-S880 specific mAb (02.22.4, made in-house), anti-GluA2 phospho- Yong for their critical reading and editing of the manuscript. This work was Y876 specific mAb (045.10.5, made in-house), anti-PSD95 mAb (NeuroMab), supported by a grant from the NIH (R01 NS036715).

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