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Extensive Phosphorylation of AMPA Receptors in Neurons

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Extensive Phosphorylation of AMPA Receptors in Neurons Extensive phosphorylation of AMPA receptors in neurons Graham H. Dieringa,1, Seok Heoa,1, Natasha K. Hussaina,1, Bian Liua, and Richard L. Huganira,b,2 aSolomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bKavli Neuroscience Discovery Institute, Johns Hopkins University, Baltimore, MD 21205 Contributed by Richard L. Huganir, July 1, 2016 (sent for review March 17, 2016; reviewed by Andres Barria and Katherine W. Roche) Regulation of AMPA receptor (AMPAR) function is a fundamental that phospho-S831 and phospho-S845 GluA1 was less than 1% or mechanism controlling synaptic strength during long-term poten- 0.1% of total GluA1, respectively (24). These findings claimed to tiation/depression and homeostatic scaling. AMPAR function and call into question the role of GluA1 phosphorylation in synaptic membrane trafficking is controlled by protein–protein interac- plasticity. Here we examined the extent of GluA1 phosphory- tions, as well as by posttranslational modifications. Phosphoryla- lation using a variety of methods. Consistent with earlier work tion of the GluA1 AMPAR subunit at S845 and S831 play especially and models (1), we find that many of excitatory synapses are important roles during synaptic plasticity. Recent controversy has positive for phosphorylated GluA1, and a large fraction of the emerged regarding the extent to which GluA1 phosphorylation total GluA1-containing receptor population is phosphorylated. may contribute to synaptic plasticity. Here we used a variety of Approximately 15–20% of GluA1-containing AMPARs are methods to measure the population of phosphorylated GluA1-con- phosphorylated at either S845 or S831 under basal conditions in taining AMPARs in cultured primary neurons and mouse forebrain. vitro and in vivo, and these levels increase to ∼50% under Phosphorylated GluA1 represents large fractions from 12% to 50% certain pharmacological or behavioral stimulation conditions. of the total population under basal and stimulated conditions in We confirm the existence of GluA1-containing receptors dually vitro and in vivo. Furthermore, a large fraction of synapses are phosphorylated at S845/S831. Thus, our findings support the positive for phospho-GluA1–containing AMPARs. Our results sup- port the large body of research indicating a prominent role of GluA1 important role of AMPAR phosphorylation in synaptic biology. phosphorylation in synaptic plasticity. Results Detection of Phospho-GluA1 Using Immunofluorescence Microscopy. synaptic plasticity | excitatory synapse | AMPA receptor | protein kinase A | – protein kinase C First, we examined the distribution of phospho-GluA1 contain- ing AMPARs at synapses using immunofluorescence micros- copy. Rat hippocampal neurons were left untreated, or treated etrameric AMPA-type glutamate receptors (AMPARs), made for 10 min with forskolin/rolipram (FR; 5 μM/100 nM) or up from the subunits GluA1–4, mediate the majority of the fast T phorbol-myristoyl-acetate (PMA; 1 μM) to selectively activate excitatory synaptic transmission in the central nervous system (1). PKA or PKC, respectively, or the noradrenaline analog, iso- Regulation of AMPAR function is highly dynamic and represents a μ fundamental mechanism to control synaptic strength for many proterenol (Iso; 5 M), followed by fixation and immunolabeling forms of synaptic plasticity, including long-term potentiation and using GluA1 and VGlut1 antibodies and phospho-specific antibodies depression (LTP/LTD) and homeostatic scaling (1). The function and membrane trafficking of AMPARs is regulated Significance by multiple interacting proteins, as well as by posttranslational modifications, including phosphorylation, palmitoylation, and Decades of research from many laboratories has established a ubiquitination (1). In particular, phosphorylation of GluA1 at model in which phosphorylation of the GluA1 AMPA-type S845 by cyclic-AMP–dependent protein kinase (PKA) (2) and glutamate receptor subunit plays a significant role modulat- at S831 by calcium/calmodulin-dependent protein kinase II ing long-term potentiation and depression, homeostatic and (CaMKII) or protein kinase C (PKC) (3, 4) have been exten- neuromodulator-regulated plasticity, spatial memory, fear/ sively studied. PKA-mediated phosphorylation of GluA1 S845 extinction, and appetitive incentive learning. However, a re- hasbeenshowntopromoteGluA1cell-surface insertion and syn- cent study suggests that GluA1 phosphorylation is exceed- aptic retention, increase channel open-probability, facilitate the ingly low, even in synaptic fractions. Here, we address this induction of LTP, and mediate homeostatic scaling-up, whereas controversy using in vitro and in vivo techniques. We find a dephosphorylation of S845 is associated with receptor endocytosis, large fraction of excitatory synapses are positive for phos- LTD, and homeostatic scaling-down (5–13). CaMKII-mediated phorylated GluA1. Moreover, phosphorylated species make phosphorylation of GluA1 S831 increases channel conductance up a significant fraction of the population and are highly re- and regulates LTP (8, 13–16). Perhaps the strongest evidence for sponsive to numerous physiologically relevant stimuli. This roles of GluA1 S845 and S831 phosphorylation comes from studies characterization reaffirms a large body of work defining a of mice with knockin mutations in which GluA1 phosphorylation is prominent role of AMPA receptor phosphorylation in synapse disrupted or mimicked. Several forms of behavior and synaptic biology and synaptic plasticity. plasticity in multiple brain regions require S845 or S831 phosphor- Author contributions: G.H.D., S.H., N.K.H., and R.L.H. designed research; G.H.D., S.H., and ylation, including hippocampal/cortical LTP and LTD, homeostatic N.K.H. performed research; G.H.D., S.H., N.K.H., and B.L. analyzed data; and G.H.D., S.H., plasticity, modulation of plasticity by neuromodulators, hippocam- N.K.H., B.L., and R.L.H. wrote the paper. pal spatial memory, fear-learning/extinction, appetitive incentive Reviewers: A.B., University of Washington; and K.W.R., National Institutes of Health, Re- learning, and the action of antidepressants (8, 10–12, 17–23). ceptor Biology Section/National Institute of Neurological Disorders and Stroke. A recent study (24) used a variant of SDS/PAGE, called Phos- The authors declare no conflict of interest. tag, to quantify the stoichiometry of GluA1 S845/S831 phosphor- 1G.H.D., S.H., and N.K.H. contributed equally to this work. ylation. Surprisingly, the authors of this study found that the levels 2To whom correspondence should be addressed. Email: [email protected]. of GluA1 phosphorylation were extremely low, even in synaptic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fractions, contrary to earlier studies (9). This recent work reports 1073/pnas.1610631113/-/DCSupplemental. E4920–E4927 | PNAS | Published online August 1, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1610631113 Downloaded by guest on September 28, 2021 A PNAS PLUS B C D NEUROSCIENCE Fig. 1. Immunofluorescence detection of synaptic phospho-S845 and S831. (A and B) Rat hippocampal neurons (14 DIV) were treated with FR, PMA, or Iso for 10 min, followed by fixation and immunolabeling. Phospho-S845 (A) or phospho-S831 (B) shows clear punctate staining that overlaps with excitatory synapse marker VGlut1. Representative images are 40-μm dendritic segments. Quantification of phospho-S845 (C) or phospho-S831 (D) staining intensity at synapses. **P < 0.001 (ANOVA followed by Fisher’s PLSD post hoc test, n = 20–22 cells per group). Error bars indicate mean ± SEM. See also Figs. S1 and S2. for S831 or S845 (Fig. 1). Synaptic phospho-GluA1 signal antiphospho-S831 in S831A neurons under any treatment condi- was identified as phospho-S845 or -S831 puncta overlapping with tions, confirming the specificity of these antibodies for immuno- both VGlut1 and total GluA1. Under basal conditions, both anti- labeling (Fig. S1). In addition, with this immunolabeling method, phospho-S845 and -S831 showed punctate staining that overlapped we conclude that phosphorylation of S845 and S831 can occur in- with total GluA1 and the excitatory synapse marker VGlut1 (Fig. 1 dependently, as mutation of one site did not prevent up-regulation A and B). Treatment with FR significantly increased the synaptic of the other site under the appropriate stimulation conditions (Fig. staining intensity of phospho-S845 (Fig. 1 A and C), whereas PMA S1). To further demonstrate the specificity of our immunolabeling, treatment significantly increased the synaptic intensity of phospho- we treated fixed rat hippocampal neurons with alkaline phospha- S831 (Fig. 1 B and D)[**P < 0.001, ANOVA followed by Fisher’s tase (CIP) before immunostaining (Fig. S2). We determined that partial least-squares difference (PLSD) post hoc test, n = 20–22 phosphatase treatment significantly reduced phospho-S845 and cells per group]. Under basal conditions, 16% and 76% (SEM ± phospho-S831 under basal as well as stimulated conditions (P < 3%, n ≥ 17 neuronal fields for each condition) of synapses were 0.0001 non-CIP vs. CIP-treated conditions, ANOVA; n ≥ 17 neu- positive for phospho-S845 and phospho-S831, respectively. FR or ronal fields for each condition). In summary, we found that a large PMA treatment increased the percentage to 87% and 85% (SEM ± fraction of synapses were positive for phospho-GluA1 species under 3%, n ≥ 17 neuronal fields for each condition),
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