This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular Loss of protein arginine methyltransferase 8 alters synapse composition and function, resulting in behavioral defects. Jay Penney1,2, Jinsoo Seo1,2, Oleg Kritskiy1,2, Sara Elmsaouri1,2, Fan Gao1,2, Ping-Chieh Pao1,2, Susan C. Su1,2 and Li-Huei Tsai1,2 1The Picower Institute for Learning and Memory 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA, 20139 DOI: 10.1523/JNEUROSCI.0591-17.2017 Received: 2 March 2017 Revised: 6 July 2017 Accepted: 25 July 2017 Published: 3 August 2017 Author contributions: J.P. and L.-H.T. designed research; J.P., J.S., O.K., S.E., P.-C.P., and S.C.S. performed research; J.P., J.S., O.K., and F.G. analyzed data; J.P. and L.-H.T. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was partially funded by the National Institutes of Health (grant 2-R37-NS051874-21 to LHT), the Belfer Neurodegeneration Consortium and a Human Frontier Science Program Long Term Fellowship to JP. We thank A. Waston and N. Dedic for insightful comments, and E. McNamara for animal maintenance. Corresponding author: Li-Huei Tsai, The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA, USA, 20139. [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0591-17.2017 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 © 2017 the authors 1 Title: Loss of protein arginine methyltransferase 8 alters synapse composition 2 and function, resulting in behavioral defects. 3 Abbreviated title: PRMT8 regulates hippocampal function and plasticity 4 Author names and affiliation: Jay Penney1,2, Jinsoo Seo1,2, Oleg Kritskiy1,2, Sara 5 Elmsaouri1,2, Fan Gao1,2, Ping-Chieh Pao1,2, Susan C. Su1,2, Li-Huei Tsai1,2. 6 1The Picower Institute for Learning and Memory, 7 2Department of Brain and Cognitive Sciences, Massachusetts Institute of 8 Technology, Cambridge, MA, USA, 20139 9 Corresponding author: Li-Huei Tsai 10 The Picower Institute for Learning and Memory 11 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 12 77 Massachusetts Ave., Cambridge, MA, USA, 20139. [email protected] 13 Number of pages: 37 14 Number of figures: 7 15 Number of words, Abstract: 138, Introduction: 431, Discussion: 1500 16 Conflict of interest: The authors declare no competing financial interests. 17 Acknowledgements: This work was partially funded by the National Institutes of 18 Health (grant 2-R37-NS051874-21 to LHT), the Belfer Neurodegeneration 19 Consortium and a Human Frontier Science Program Long Term Fellowship to JP. We 20 thank A. Waston and N. Dedic for insightful comments, and E. McNamara for animal 21 maintenance. 22 1 23 Abstract 24 Diverse molecular mechanisms regulate synaptic composition and function in the 25 mammalian nervous system. The multifunctional protein arginine 26 methyltransferase 8 (PRMT8) possesses both methyltransferase and phospholipase 27 activities. Here we examine the role of this neuron-specific protein in hippocampal 28 plasticity and cognitive function. PRMT8 protein localizes to synaptic sites and 29 conditional whole brain Prmt8 deletion results in altered levels of multiple synaptic 30 proteins in the hippocampus, using both male and female mice. Interestingly these 31 altered protein levels are due to post-transcriptional mechanisms as the 32 corresponding mRNA levels are unaffected. Strikingly, electrophysiological 33 recordings from hippocampal slices of mice lacking PRMT8 reveal multiple defects 34 in excitatory synaptic function and plasticity. Furthermore, behavioral analyses 35 show that PRMT8 conditional knockout mice exhibit impaired hippocampal- 36 dependent fear learning. Together these findings establish PRMT8 as an important 37 component of the molecular machinery required for hippocampal neuronal 38 function. 39 40 Significance Statement 41 Numerous molecular processes are critically required for normal brain function. 42 Here we use mice lacking protein arginine methyltransferase 8 (PRMT8) in the 43 brain to examine how loss of this protein affects the structure and function of 44 neurons in the hippocampus. We find that PRMT8 localizes to the sites of 45 communication between neurons. Hippocampal neurons from mice lacking PRMT8 2 46 have no detectable structural differences compared to controls; however, multiple 47 aspects of their function are altered. Consistently we find that mice lacking PRMT8 48 also exhibit reduced hippocampus-dependent memory. Together our findings 49 establish important roles for PRMT8 in regulating neuron function and cognition in 50 the mammalian brain. 51 52 Introduction 53 A myriad of molecular mechanisms regulate synaptic plasticity and cognitive 54 processes in the mammalian brain. The regulation of gene expression, mRNA 55 trafficking to synaptic sites and numerous post-translational mechanisms including 56 protein trafficking, phosphorylation, ubiquitination and acetylation are all critical to 57 neuronal function (Kandel, 2001). The protein arginine methyltransferase (PRMT) 58 family of enzymes regulates numerous cellular processes and pathways; however 59 the roles of these proteins in brain function have only begun to be explored 60 (Bedford and Clarke, 2009). 61 62 PRMT8 is unique among the PRMT proteins in that it is expressed specifically in the 63 nervous system (Lee et al., 2005; Kousaka et al., 2009). Furthermore, PRMT8 is a 64 multifunctional protein, exhibiting both arginine methyltransferase and 65 phospholipase D activities (Lee et al., 2005; Kim et al., 2015). Phospholipase D 66 enzymes play important roles in nervous system development and function, while 67 the identification of many arginine-methylated synaptic proteins suggests that this 68 modification may also be an important regulator of synapse biology (Klein, 2005; 3 69 Burkhardt et al., 2014; Guo et al., 2014). PRMTs, including PRMT8, have been 70 ascribed nuclear roles in regulating gene expression; however PRMT8 protein can 71 be myristoylated and membrane-targeted, suggesting it could localize to synaptic 72 sites as well (Lee et al., 2005; Bedford and Clarke, 2009; Simandi et al., 2015; Lee et 73 al., 2017). 74 75 The zebrafish PRMT8 ortholog was found to be important for nervous system 76 development, while mouse studies suggest roles for PRMT8 in cerebellar granule 77 neurons, as well as in GABAergic interneurons of the visual cortex (Kim et al., 2015; 78 Lee et al., 2017). Kim et al. (2015) showed that conditional Prmt8 deletion inhibited 79 the arborization of granule cells and reduced acetylcholine levels in the cerebellum. 80 More recently, Lee et al. (2017) found that Prmt8 mutation affected perineuronal 81 net formation in the visual cortex, as well as visual acuity of mutant mice. Thus far 82 the potential effects of PRMT8 on synaptic function and cognitive processes have 83 not been explored. 84 85 Here, we examine the roles of PRMT8 in hippocampal neuronal morphology, 86 composition and function. We find that PRMT8 protein localizes to pre- and post- 87 synaptic sites, but that loss of PRMT8 does not affect synapse density, or dendritic 88 spine density or morphology. PRMT8 conditional deletion with Nestin-cre does, 89 however, alter levels of a number of synaptic proteins and affect multiple measures 90 of synaptic function and plasticity. Further, while PRMT8 mutation does not 91 significantly alter locomotor or anxiety-related behaviors, context-dependent fear 4 92 learning is impaired in Prmt8 conditional knockout mice. Together these findings 93 establish novel roles for PRMT8 in regulating excitatory synapse composition and 94 function as well as cognitive processes, in the mammalian nervous system. 95 96 Materials and Methods 97 Mice: All experiments were performed according to the Guide for the Care and Use 98 of Laboratory Animals and were approved by the National Institutes of Health and 99 the Committee on Animal Care at the Massachusetts Institute of Technology. 100 Prmt8tm1a(EUCOMM)Wtsi (Skarnes et al., 2011) embryos were obtained from the 101 European Mutant Mouse Consortium. Following re-derivation the mice were 102 crossed to FLP recombinase expressing mice (Vooijs et al., 1998) to generate mice 103 with the Prmt8 5th exon flanked by loxP sites. Following intercrosses to generate 104 homozygous Prmt8 floxed mice they were bred to Nestin-cre expressing mice 105 (Tronche et al., 1999) to conditionally delete Prmt8 exon 5. Prmt8 floxed/Prmt8 106 floxed mice served as controls, while experimental mice were Prmt8 floxed/Prmt8 107 floxed; Nestin-cre/+ (referred to as Prmt8 cKO). 10-14 week old mice were used for 108 all experiments except for mIPSC and GluN2A-mediated recordings. Mice for these 109 latter experiments were 16-18 weeks old. Male mice were used for immunostaining, 110 Golgi staining, electrophysiology and behavior experiments. Both male and female 111 mice were used for qPCR and biochemistry experiments. 112 113 Primary neuronal culture: Primary forebrain neurons were cultured from E16.5 114 Swiss Webster embryos and analyzed at DIV17. Briefly, forebrains were removed, 5 115 digested with Papain and plated on poly-D-lysine (PDL; Sigma) coated glass 116 coverslips for immunostaining (100K cells per well in 24-well plates) or PDL coated 117 tissue-culture plates (3M cells per 10 cm plate) for cell fractionation experiments. 118 119 Cloning: The Prmt8 transcript was amplified from mouse hippocampal cDNA using 120 the primers F: 5’-TGCTCTAGAGCAGAAGTTGGGAGAGTTGC-3’ and R: 5’- 121 AGAGCGATCGCACGCATTTTGTAGTCATT-3’. The amplified region spanned from 122 base pairs 29 to 1594 of the Prmt8 transcript (NM_201371.2) including the coding 123 sequence and most of the 5’UTR (to mimic in vivo start codon selection to the extent 124 possible).