This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase Regulation of Ca2+-Permeable AMPA Receptors Jennifer L. Sanderson1, John D. Scott3,4 and Mark L. Dell'Acqua1,2 1Department of Pharmacology 2Program in Neuroscience, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO 80045, USA. 3Howard Hughes Medical Institute 4Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA. DOI: 10.1523/JNEUROSCI.2362-17.2018 Received: 21 August 2017 Revised: 17 January 2018 Accepted: 6 February 2018 Published: 13 February 2018 Author contributions: J.S., J.D.S., and M.L.D. designed research; J.S. performed research; J.S. and M.L.D. analyzed data; J.S., J.D.S., and M.L.D. wrote the paper; J.D.S. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. This work was supported by NIH grant R01NS040701 (to M.L.D.). Contents are the authors' sole responsibility and do not necessarily represent official NIH views. The authors declare no competing financial interests. We thank Dr. Jason Aoto for critical reading of the manuscript. Corresponding author: Mark L. Dell'Acqua, Department of Pharmacology, University of Colorado School of Medicine, Mail Stop 8303, RC1-North, 12800 East 19th Ave Aurora, CO 80045. Email: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.2362-17.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Title: Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase 2 Regulation of Ca2+-Permeable AMPA Receptors 3 4 Abbreviated title: AKAP150 regulation of homeostatic synaptic scaling 5 6 Authors: Jennifer L. Sanderson1, John D. Scott3,4 and Mark L. Dell’Acqua1,2 7 Affiliations: 1Department of Pharmacology and 2Program in Neuroscience, University of 8 Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO 80045, USA. 3Howard 9 Hughes Medical Institute and 4Department of Pharmacology, University of Washington School 10 of Medicine, Seattle, WA 98195, USA. 11 Corresponding author: Mark L. Dell’Acqua, Department of Pharmacology, University of 12 Colorado School of Medicine, Mail Stop 8303, RC1-North, 13 12800 East 19th Ave Aurora, CO 80045. Email: [email protected] 14 15 Number of pages: 43 16 Number of Figures: 5 17 Abstract: 249 words 18 Significance statement: 119 words 19 Introduction: 646 words 20 Discussion: 1499 words 21 22 Acknowledgements: This work was supported by NIH grant R01NS040701 (to M.L.D.). 23 Contents are the authors’ sole responsibility and do not necessarily represent official NIH views. 24 The authors declare no competing financial interests. We thank Dr. Jason Aoto for critical 25 reading of the manuscript. 26 1 27 Abstract: Neuronal information processing requires multiple forms of synaptic plasticity 28 mediated by NMDA and AMPA-type glutamate receptors (NMDAR, AMPAR). These plasticity 29 mechanisms include long-term potentiation (LTP) and depression (LTD), which are Hebbian, 30 homosynaptic mechanisms locally regulating synaptic strength of specific inputs, and 31 homeostatic synaptic scaling, which is a heterosynaptic mechanism globally regulating synaptic 32 strength across all inputs. In many cases, LTP and homeostatic scaling regulate AMPAR 33 subunit composition to increase synaptic strength via incorporation of Ca2+-permeable receptors 34 (CP-AMPAR) containing GluA1, but lacking GluA2, subunits. Previous work by our group and 35 others demonstrated that anchoring of the kinase PKA and the phosphatase calcineurin (CaN) 36 to A-kinase anchoring protein (AKAP) 150 play opposing roles in regulation of GluA1 Ser845 37 phosphorylation and CP-AMPAR synaptic incorporation during hippocampal LTP and LTD. Here, 38 using both male and female knock-in mice that are deficient in PKA or CaN anchoring, we show 39 that AKAP150-anchored PKA and CaN also play novel roles in controlling CP-AMPAR synaptic 40 incorporation during homeostatic plasticity in hippocampal neurons. We found that genetic 41 disruption of AKAP-PKA anchoring prevented increases in Ser845 phosphorylation and CP- 42 AMPAR synaptic recruitment during rapid homeostatic synaptic scaling-up induced by combined 43 blockade of action potential firing and NMDAR activity. In contrast, genetic disruption of AKAP- 44 CaN anchoring resulted in basal increases in Ser845 phosphorylation and CP-AMPAR synaptic 45 activity that blocked subsequent scaling-up by preventing additional CP-AMPAR recruitment. 46 Thus, the balanced, opposing phospho-regulation provided by AKAP-anchored PKA and CaN is 47 essential for control of both Hebbian and homeostatic plasticity mechanisms that require CP- 48 AMPARs. 49 50 2 51 Significance statement: Neuronal circuit function is shaped by multiple forms of activity- 52 dependent plasticity that control excitatory synaptic strength, including LTP/LTD that adjusts 53 strength of individual synapses and homeostatic plasticity that adjusts overall strength of all 54 synapses. Mechanisms controlling LTP/LTD and homeostatic plasticity were originally thought 55 to be distinct; however, recent studies suggest that CP-AMPAR phosphorylation regulation is 56 important during both LTP/LTD and homeostatic plasticity. Here we show that CP-AMPAR 57 regulation by the kinase PKA and phosphatase CaN co-anchored to the scaffold protein 58 AKAP150, a mechanism previously implicated in LTP/LTD, is also crucial for controlling synaptic 59 strength during homeostatic plasticity. These novel findings significantly expand our 60 understanding of homeostatic plasticity mechanisms and further emphasize how intertwined 61 they are with LTP and LTD. 62 63 3 64 Introduction 65 Hebbian plasticity in the hippocampus and cortex is input-specific, bi-directional 66 (LTP/LTD), and can be induced rapidly (seconds-minutes) but expressed persistently (hours- 67 days)(Collingridge et al., 2010; Huganir and Nicoll, 2013). However, excitatory neurons in these 68 two regions can also persistently scale-up or -down synaptic strength across all inputs in 69 response to chronic (hours-days) decreases or increases, respectively, in overall input and firing. 70 This global compensatory adjustment of synaptic strength is termed homeostatic synaptic 71 plasticity and is thought to stabilize neuronal circuits by constraining firing and synaptic strength 72 within ranges that maintain connectivity, while preventing hyper-excitability and preserving 73 Hebbian plasticity (Turrigiano, 2012; Chen et al., 2013; Lee et al., 2013). Not surprisingly, 74 alterations in both LTP/LTD and homeostatic plasticity are implicated in nervous system 75 diseases (Wondolowski and Dickman, 2013). Thus, it is of great interest to understand how 76 Hebbian and homeostatic plasticity interact (Keck et al., 2017). 77 Homeostatic plasticity, like Hebbian, can be induced by changes in Ca2+ signaling 78 downstream of NMDARs and/or L-type voltage-gated Ca2+ channels and are both expressed 79 through changes in AMPAR localization and activity (O'Brien et al., 1998; Turrigiano et al., 80 1998; Thiagarajan et al., 2005; Sutton et al., 2006; Aoto et al., 2008; Ibata et al., 2008; Lee and 81 Chung, 2014). Nonetheless, it was originally thought that downstream mechanisms mediating 82 Hebbian and homeostatic AMPAR regulation would be largely non-overlapping. However, 83 accumulating evidence indicates that these processes utilize many of the same signaling 84 pathways. Indeed, several studies have implicated ser/thr protein kinases and phosphatases in 85 homeostatic plasticity that also have well characterized roles in LTP/LTD, including Ca2+-CaM- 86 dependent protein kinase II (CaMKII)(Thiagarajan et al., 2002; Thiagarajan et al., 2005; Groth et 87 al., 2011), the cAMP-dependent protein kinase PKA (Goel et al., 2011; Diering et al., 2014), and 88 the Ca2+-CaM-dependent protein phosphatase 2B/CaN (Kim and Ziff, 2014). These postsynaptic 89 kinase/phosphatase signaling pathways may regulate synaptic strength during both LTP/LTD 4 90 and homeostatic plasticity at least in part through regulating the synaptic incorporation of high- 91 conductance GluA1 CP-AMPARs (Thiagarajan et al., 2005; Plant et al., 2006; Lu et al., 2007; 92 Yang et al., 2010; Goel et al., 2011; Soares et al., 2013; Kim and Ziff, 2014; Sanderson et al., 93 2016), which are largely excluded from hippocampal synapses under basal conditions (Lu et al., 94 2009; Rozov et al., 2012). In addition, homeostatic synaptic plasticity, like LTP/LTD, can be 95 induced and expressed at individual synapses in dendrites through local regulation of protein 96 synthesis and GluA1 CP-AMPAR delivery, further suggesting common underlying mechanisms 97 (Sutton et al., 2006; Hou et al., 2008; Maghsoodi et al., 2008; Soares et al., 2013). 98 Our laboratory and others have shown that PKA and CaN anchored to the scaffold 99 AKAP79/150 (79 human/150 rodent; Akap5 gene) play key opposing roles regulating GluA1 100 Ser845 phosphorylation to control CP-AMPAR recruitment/removal at hippocampal synapses 101 during LTP/LTD (Lu et al., 2007; Sanderson et al., 2012;