This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease AMPA receptor dysregulation and therapeutic interventions in a mouse model of CDKL5 Deficiency Disorder Madhumita Yennawar1, Rachel S. White, PhD2 and Frances E. Jensen, MD2 1Department of Systems Pharmacology and Translational Therapeutics 2Department of Neurology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104 https://doi.org/10.1523/JNEUROSCI.2041-18.2019 Received: 9 August 2018 Revised: 13 March 2019 Accepted: 27 March 2019 Published: 5 April 2019 Author contributions: M.Y. and F.E.J. designed research; M.Y. and R.S.W. performed research; M.Y. analyzed data; M.Y. wrote the first draft of the paper; M.Y., R.S.W., and F.E.J. edited the paper; M.Y. and F.E.J. wrote the paper; F.E.J. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. This work was supported by NIH grants NS 031718 (FEJ), NS 080565 (FEJ), DP1 OD003347 (FEJ), Hope4Harper Foundation, and the Lou Lou Foundation (MY and FEJ). We would like to acknowledge the Neurobehavior Testing Core at UPenn and IDDRC at CHOP/Penn U54 HD086984 for assistance with behavior procedures. We would also like to thank Dr. Zhaolan Zhou, Professor of Genetics, University of Pennsylvania, for his guidance and Dr. Delia Talos, Assistant Professor of Neurology, University of Pennsylvania, for advice with human tissue analysis. We also thank Dr. Chengwen Zhou, Assistant Professor of Neurology, Vanderbilt University for his help with electrophysiology analysis and Sydney Zebrowitz for her work assessing animal behavior. Correspondence should be sent to: Frances E. Jensen, MD, FACP, Professor and Chair, Department of Neurology, Co-Director Penn Medicine Translational Neuroscience Program, Perelman School of Medicine, University of Pennsylvania, 3400 Spruce Street, 3rd Floor Dulles Building, Philadelphia, PA 19104-4283, Phone (215) 662-3360, Email [email protected] Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.2041-18.2019 Alerts: Sign up at www.jneurosci.org/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 © 2019 the authors 1 Title: AMPA receptor dysregulation and therapeutic interventions in a mouse model of 2 CDKL5 Deficiency Disorder 3 4 Madhumita Yennawar1, Rachel S. White, PhD2, Frances E. Jensen, MD2 5 1Department of Systems Pharmacology and Translational Therapeutics and 2Department of 6 Neurology, University of Pennsylvania Perelman School of Medicine, Philadelphia, 7 Pennsylvania 19104 8 9 Correspondence should be sent to: 10 Frances E. Jensen, MD, FACP 11 Professor and Chair 12 Department of Neurology 13 Co-Director Penn Medicine Translational Neuroscience Program 14 Perelman School of Medicine, University of Pennsylvania 15 3400 Spruce Street, 3rd Floor Dulles Building 16 Philadelphia, PA 19104-4283 17 Phone (215) 662-3360 18 Email [email protected] 19 20 Number of pages: 35 21 Number of figures: 9 figures 22 Number of words in abstract: 191 23 Number of words in introduction: 648 24 Number of words in discussion: 1493 25 Conflict of interest: The authors declare no competing financial interests. 26 Acknowledgements: This work was supported by NIH grants NS 031718 (FEJ), NS 080565 27 (FEJ), DP1 OD003347 (FEJ), Hope4Harper Foundation, and the Lou Lou Foundation (MY and 28 FEJ). We would like to acknowledge the Neurobehavior Testing Core at UPenn and IDDRC at 29 CHOP/Penn U54 HD086984 for assistance with behavior procedures. We would also like to 30 thank Dr. Zhaolan Zhou, Professor of Genetics, University of Pennsylvania, for his guidance and 31 Dr. Delia Talos, Assistant Professor of Neurology, University of Pennsylvania, for advice with 32 human tissue analysis. We also thank Dr. Chengwen Zhou, Assistant Professor of Neurology, 33 Vanderbilt University for his help with electrophysiology analysis and Sydney Zebrowitz for her 34 work assessing animal behavior. 35 36 1 37 Abstract: 38 Pathogenic mutations in cyclin-dependent kinase-like 5 (CDKL5) result in CDKL5 Deficiency 39 Disorder (CDD), a rare disease marked by early-life seizures, autistic behaviors, and intellectual 40 disability. Although mouse models of CDD exhibit dendritic instability and alterations in synaptic 41 scaffolding proteins, studies of glutamate receptor levels and function are limited. Here we used 42 a novel mouse model of CDD, the Cdkl5R59X knock-in mouse (R59X), to investigate changes in 43 synaptic glutamate receptor subunits and functional consequences. Male mice were used for all 44 experiments to avoid the confounding effects of X-inactivation that would be present in female 45 heterozygous mice. We showed that adult male R59X mice recapitulated the behavioral 46 outcomes observed in other mouse models of CDD, including social deficits and memory and 47 learning impairments, and exhibited decreased latency to seizure upon pentylenetetrazol (PTZ) 48 administration. Furthermore, we observed a specific increase in GluA2-lacking AMPA receptors 49 (AMPARs) in the adult R59X hippocampus, which is accompanied electrophysiologically by 50 increased rectification ratio of AMPAR excitatory post-synaptic currents and elevated early- 51 phase long-term potentiation. Finally, we showed that acute treatment with the GluA2-lacking 52 AMPAR blocker IEM-1460 decreased AMPAR currents, and rescued social deficits, working 53 memory impairments, and seizure behavior latency in R59X mice. 54 55 Significance Statement: CDKL5 Deficiency Disorder (CDD) is a rare disease marked by 56 autistic-like behaviors, intellectual disability, and seizures. While synaptic dysfunction has been 57 observed in mouse models of CDD, there is limited information on how synaptic alterations 58 contribute to behavioral and functional changes in CDD. Here we reveal elevated hippocampal 59 GluA2-lacking AMPAR expression in a novel mouse model of CDD that is accompanied by 60 changes in synaptic AMPAR function and plasticity. We also show for the first time that acutely 61 targeting GluA2-lacking AMPAR dysregulation rescues core synaptic and neurobehavioral 62 deficits in CDD. 2 63 3 64 Introduction 65 CDKL5 Deficiency Disorder (CDD) is a severe neurological disease caused by pathogenic 66 mutations in the X-linked gene for cyclin-dependent kinase-like 5 (CDKL5), a serine-threonine 67 kinase that is highly expressed in the brain (Montini et al., 1998; Kilstrup-Nielsen et al., 2012). 68 Patients with CDD exhibit intellectual disability (ID), autistic-like behaviors, and early-life 69 epilepsy, neurological deficits that often co-occur and share underlying mechanisms of synaptic 70 dysfunction. Indeed, mouse models of CDD exhibit structural and functional changes at 71 excitatory dendritic spines, which are accompanied by behavioral alterations such as social 72 interaction deficits, and impaired learning and memory (Wang et al., 2012; Amendola et al., 73 2014; et al., 2017; Tang et al., 2017). Furthermore, CDKL5 itself has been found to localize to 74 synapses and interact with postsynaptic density protein 95 (PSD-95), netrin-G1 ligand (NGL-1) 75 and Rac1 (Chen et al., 2010; Ricciardi et al., 2012; Zhu et al., 2013)— proteins that are involved 76 with clustering and transport of excitatory glutamate receptors at the synapse. However, there is 77 limited information regarding the link between alterations in synaptic glutamate receptors and 78 neurobehavioral deficits in CDD mouse models. 79 80 Glutamate receptors, which include AMPA, NMDA, and kainate receptors, are heteromeric 81 structures that mediate excitatory neurotransmission in the brain. Receptor subunits are 82 dynamically expressed, altering receptor composition and biophysical properties to regulate the 83 excitatory-inhibitory (E-I) balance throughout the lifetime (Luján et al., 2005; Rakhade and 84 Jensen, 2009). Dysregulated glutamate receptor subunit expression is often associated with 85 epilepsy, autism, and ID (Loddenkemper et al., 2014; Mignogna et al., 2015; Lippman-Bell et 86 al., 2016). In particular, even modest changes in the tightly regulated GluA2 AMPAR subunit 87 have a profound effect on excitability and signal transduction due to the distinct properties of 88 GluA2 (Lippman-Bell et al., 2013; Uzunova et al., 2014; Stephenson et al., 2017). Most notably, 89 GluA2-lacking AMPARs are Ca2+-permeable (CP-AMPARs) and play a significant role in 4 90 regulating dendritic spine enlargement as well as downstream signaling and gene transcription 91 (Plant et al., 2006; et al., 2007; Fortin et al., 2010; Henley and Wilkinson, 2016). Indeed, we 92 have previously observed decreased GluA2 expression following neonatal hypoxic seizures in 93 wild-type rats, which contributes to social interaction deficits, elevated activity-dependent Ca2+ 94 influx, altered downstream signaling, and increased epileptogenesis (Sanchez et al., 2001; 95 Rakhade et al., 2008; Lippman-Bell et al., 2013, 2016; Rosenberg et al., 2018). Finally, altered 96 GluA2 subunit expression has been observed in several neurodevelopmental disorders such as 97 Rett syndrome, Fragile X syndrome, tuberous sclerosis, and RAB39B-associated mental 98 retardation (Talos et al., 2008; Mignogna et al., 2015; Li et al., 2016; Achuta et al., 2018), 99 pointing