Research Articles: Cellular/Molecular The mRNA-binding protein RBM3 regulates activity patterns and local synaptic translation in cultured hippocampal neurons https://doi.org/10.1523/JNEUROSCI.0921-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0921-20.2020 Received: 20 April 2020 Revised: 14 October 2020 Accepted: 12 November 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Sertel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 The mRNA-binding protein RBM3 regulates activity patterns and local synaptic 2 translation in cultured hippocampal neurons 3 4 Abbreviated title: RBM3 regulates neuronal activity and translation 5 6 Sinem M. Sertel1*, Malena S. von Elling-Tammen1, Silvio O. Rizzoli1,2* 7 8 1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, 9 Göttingen, 37073, Germany 10 2Lead Contact 11 *Correspondence: [email protected] (S.M.S.), [email protected] (S.O.R.) Number of pages 43 (with 2.0 spacing) Number of figures 10 Number of tables 1 Number of words in abstract 163 Number of words in introduction 658 Number of words in discussion 1317 12 13 Competing interests 14 The authors declare that they have no competing interests. 15 Acknowledgements 16 We would like to thank Verena Klüver and Dr. Eugenio Fornasiero for their generous gift of 17 the Scr shRNA plasmid, Roya Yousefi and Prof. Peter Rehling for their generous gift of 18 puromycin, anisomycin and puromycin antibody, and Janina Pasch for her help throughout 19 the puromycin experiments (all from University Medical Center Göttingen). We also would 20 like to thank the Transcriptome and Genome Analysis Laboratory (TAL, Göttingen, Germany) 21 for mRNA sequencing and analysis. We thank Prof. Dr. Dörthe Katschinski (University 22 Medical Center Göttingen) for access to viral production facilities. We thank Prof. Christopher 23 S. Colwell (Brain Research Institute, University of California, Los Angeles) for comments on 24 the manuscript. The work was supported by a grant from the Deutsche 25 Forschungsgemeinschaft (DFG) to S.O.R., SFB1286/A03. 1 26 The mRNA-binding protein RBM3 regulates activity patterns and local synaptic 27 translation in cultured hippocampal neurons 28 29 Sinem M. Sertel1*, Malena S. von Elling-Tammen1, Silvio O. Rizzoli1,2* 30 31 1Institute for Neuro- and Sensory Physiology, University Medical Center Göttingen, 32 Göttingen, 37073, Germany 33 2Lead Contact 34 *Correspondence: [email protected] (S.M.S.), [email protected] (S.O.R.) 35 Abstract 36 The activity and the metabolism of the brain change rhythmically during the day/night cycle. 37 Such rhythmicity is also observed in cultured neurons from the suprachiasmatic nucleus, 38 which is a critical center in rhythm maintenance. However, this issue has not been 39 extensively studied in cultures from areas less involved in timekeeping, as the hippocampus. 40 Using neurons cultured from the hippocampi of newborn rats (both male and female), we 41 observed significant time-dependent changes in global activity, in synaptic vesicle dynamics, 42 in synapse size, and in synaptic mRNA amounts. A transcriptome analysis of the neurons, 43 performed at different times over 24 hours, revealed significant changes only for RNA- 44 binding motif 3 (Rbm3). RBM3 amounts changed especially in synapses. RBM3 knock-down 45 altered synaptic vesicle dynamics and changed the neuronal activity patterns. This procedure 46 also altered local translation in synapses, albeit it left the global cellular translation 47 unaffected. We conclude that hippocampal cultured neurons can exhibit strong changes in 48 their activity levels over 24 hours, in an RBM3-dependent fashion. 49 Significance Statement 50 This work is important in several ways. First, the discovery of relatively regular activity 51 patterns in hippocampal cultures implies that future studies using this common model will 52 need to take the time parameter into account, to avoid misinterpretation. Second, our work 1 53 links these changes in activity strongly to RBM3, in a fashion that is independent of the 54 canonical clock mechanisms, which is a very surprising observation. Third, we describe here 55 probably the first molecule (RBM3) whose manipulation affects translation specifically in 56 synapses, and not at the whole-cell level. This is a key finding for the rapidly growing field of 57 local synaptic translation. 58 Introduction 59 Maintaining a synchronous pattern of day and night activity is critical for the function of all of 60 the tissues of a mammalian organism. This is ensured by several well-established 61 mechanisms, the first of which is the rhythmic expression of molecular clock genes in every 62 cell throughout the day/night (Partch et al., 2014). These genes control the timing of many 63 biological functions, such as glucose metabolism and electrical activity (Dibner et al., 2010). 64 A second fundamental mechanism is provided by the function of the suprachiasmatic 65 nucleus (SCN), a central pacemaker of the hypothalamus, which is in charge of the 66 molecular clock synchronization among the cells of the animal (Welsh et al., 2010). The SCN 67 achieves this by encoding time information in its spontaneous firing rate (low during the night, 68 and high during the day (Colwell, 2011)), and by communicating this to other brain regions 69 and tissues through synaptic projections and hormones (Buijs et al., 2006). 70 The rhythmic expression of clock genes in the SCN controls the expression and function of 71 ion channels as the BK channels (large-conductance calcium-activated potassium channels) 72 or L-type voltage-gated calcium channels (Colwell, 2011). The function of these proteins 73 induces oscillations in the resting membrane potential (Pennartz et al., 2002; Kononenko et 74 al., 2008), thereby changing the firing rates, which in turn ensures the rhythmic firing activity 75 of the SCN, which has been demonstrated even in dispersed cultures (Green and Gillette, 76 1982; Herzog et al., 1998). The rhythmic firing is resistant to disturbances in the light-dark 77 cycle (Kuhlman and McMahon, 2004; Nakamura et al., 2011), and it persists in SCN cultures 78 that are not subjected to day/night light or temperature changes. However, clock gene 79 expression alone is not sufficient to maintain the synchronized firing of SCN neurons in the 2 80 long term. In culture, they slowly become desynchronized, with every cell eventually 81 assuming its own individual firing pattern that oscillates throughout the day and night cycle 82 (Welsh et al., 1995). The desynchronization is accelerated by blocking network activity, 83 suggesting that neuronal communication is important in maintaining the rhythm synchronicity 84 for long time intervals (Honma et al., 2000; Yamaguchi et al., 2003). 85 The observation of rhythmic activity in dispersed SCN cultures prompted research also in 86 other cell types. Fibroblast cell lines were found to exhibit molecular clock rhythmicity, albeit 87 they lose cell synchronicity rapidly (Nagoshi et al., 2004), unless they are re-synchronized by 88 regular changes in temperature (Brown et al., 2002) or culture media (Balsalobre et al., 89 1998). However, many brain areas have been little investigated in relation to rhythmic activity 90 (Paul et al., 2019). A prominent example is the hippocampus, which is involved in learning 91 and memory, two processes that are strongly regulated by the circadian clock (Gerstner and 92 Yin, 2010). Hippocampal activity in vivo oscillates throughout the day and night cycle (Munn 93 and Bilkey, 2012), and its ability to respond to plasticity-inducing stimuli is also dependent on 94 the time of day/night (Harris and Teyler, 1983). This demonstrates that the hippocampus 95 function is governed by the 24 hours cycle, but leave open the question of whether this is 96 exclusively due to the general rhythmicity induced by the SCN, or whether this is a 97 fundamental hallmark of the hippocampal neuron, which would persist in dissociated 98 cultures. 99 To solve this question, we turned to the rat hippocampal culture. Surprisingly, we found that 100 the culture activity exhibited significant oscillations throughout 24 hours, which were 101 accompanied by substantial changes in presynaptic activity and synapse size. In addition, we 102 found that the abundance of RNA-binding motif 3 (Rbm3), a cold-shock protein (Danno et al., 103 1997, 2000) that is known to promote translation (Dresios et al., 2005), also oscillates 104 throughout 24 hours, especially in synapses. Its knock-down changed the activity pattern of 105 the neurons, as well as synapse activity and size, possibly through effects on local 106 translation. Overall, these data suggest that hippocampal cultures exhibit endogenous 3 107 changes in activity levels across 24 hours, and that these patterns are under the control of 108 RBM3. 109 Materials and Methods 110 Hippocampal cultures. Primary disassociated hippocampal cultures were prepared from 111 newborn rats (Banker and Cowan, 1977). The hippocampi were dissected from rat brains, 112 using animals of both sexes, with a general female to male ratio of 1:1. They were washed 113 with Hank’s balanced salt solution (HBSS, Thermo Fisher, US). Later on, hippocampi were 114 kept in the enzyme solution (1.6 mM cysteine, 100 mM CaCl2, 50 mM EDTA, and 25 units 115 papain in 10 ml Dulbecco's modified eagle medium (DMEM)) for 1 hour.