Calmodulin Suppresses Synaptotagmin-2 Transcription In
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Supplemental Material can be found at: http://www.jbc.org/content/suppl/2010/09/08/M110.150151.DC1.html THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 44, pp. 33930–33939, October 29, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons*□S Received for publication, June 1, 2010, and in revised form, July 23, 2010 Published, JBC Papers in Press, August 20, 2010, DOI 10.1074/jbc.M110.150151 Zhiping P. Pang‡1,2, Wei Xu‡§1, Peng Cao§, and Thomas C. Su¨dhof‡§3 From the ‡Department of Molecular and Cellular Physiology and the §Howard Hughes Medical Institute, Stanford University, Palo Alto, California 94304-5543 ؉ Calmodulin (CaM) is a ubiquitous Ca2 sensor protein that modes of synaptic vesicle exocytosis are triggered by Ca2ϩ. The plays a pivotal role in regulating innumerable neuronal func- synchronous release mode exhibits an apparent Ca2ϩ cooper- tions, including synaptic transmission. In cortical neurons, ativity of ϳ5 (1–3), and the asynchronous release shows an ؉ most neurotransmitter release is triggered by Ca2 binding to apparent Ca2ϩ cooperativity of ϳ2 (3). The role of synaptotag- synaptotagmin-1; however, a second delayed phase of release, mins as primary Ca2ϩ sensors for synchronous neurotransmit- ؉ referred to as asynchronous release, is triggered by Ca2 binding ter release is well established (4–11). However, the molecular ؉ Downloaded from to an unidentified secondary Ca2 sensor. To test whether CaM identity of the Ca2ϩ sensor that mediates asynchronous release ؉ could be the enigmatic Ca2 sensor for asynchronous release, remains unknown. we now use in cultured neurons short hairpin RNAs that sup- Calmodulin (CaM)4 is a ubiquitous and essential Ca2ϩ-bind- press expression of ϳ70% of all neuronal CaM isoforms. Sur- ing protein that regulates a plethora of cellular processes, from prisingly, we found that in synaptotagmin-1 knock-out neurons, gene transcription to signal transduction to ion channels to the CaM knockdown caused a paradoxical rescue of synchro- membrane traffic (12–14). CaM is highly conserved in verte- www.jbc.org nous release, instead of a block of asynchronous release. Gene brates and is ubiquitously expressed. All CaM proteins are and protein expression studies revealed that both in wild-type composed of two lobes (i.e. the N- and C-lobes) that each con- and in synaptotagmin-1 knock-out neurons, the CaM knock- tain two E-F hand Ca2ϩ-binding motifs and are connected via a 2ϩ down altered expression of >200 genes, including that encoding flexible ␣-helix (15). Each E-F hand motif binds to one Ca at UMDNJ RW JOHNSON, on June 4, 2012 synaptotagmin-2. Synaptotagmin-2 expression was increased ion. Ca2ϩ binds to the N- and C-lobes in a cooperative manner, several-fold by the CaM knockdown, which accounted for the with the N-lobe binding Ca2ϩ with a lower affinity but faster paradoxical rescue of synchronous release in synaptotagmin-1 association and dissociation rates than the C-lobe (16, 17). The knock-out neurons by the CaM knockdown. Interestingly, the different Ca2ϩ binding characteristics probably confer onto CaM knockdown primarily activated genes that are preferen- CaM lobes specific target protein binding properties and func- tially expressed in caudal brain regions, whereas it repressed tions (18, 19). Apart from numerous cytoplasmic regulatory genes in rostral brain regions. Consistent with this correlation, functions, Ca2ϩ binding to CaM serves to activate transcription quantifications of protein levels in adult mice uncovered an by a number of distinct signaling pathways (14, 20). inverse relationship of CaM and synaptotagmin-2 levels in CaM regulates neurotransmitter release by multiple mecha- mouse forebrain, brain stem, and spinal cord. Finally, we nisms, including binding to Munc13, regulating Ca2ϩ channels, employed molecular replacement experiments using a knock- and activating Ca2ϩ/CaM-dependent kinase II (CaMKII) (12– ؉ down rescue approach to show that Ca2 binding to the C-lobe 14, 20–24). In addition, CaM was proposed to directly function but not the N-lobe of CaM is required for suppression of synap- asaCa2ϩ sensor for Ca2ϩ-triggered exocytosis (25, 26), totagmin-2 expression in cortical neurons. Our data describe a prompting us to test here whether CaM may act as the Ca2ϩ ؉ previously unknown, Ca2 /CaM-dependent regulatory path- sensor for asynchronous release. For this purpose, we cultured way that controls the expression of synaptic proteins in the ros- cortical neurons from synaptotagmin-1 (Syt1) knock-out (KO) tral-caudal neuraxis. mice in which synchronous release is abolished and only asyn- chronous release remains (5, 6). We then analyzed the effects of shRNA-mediated knockdown (KD) of all CaM isoforms on Neurotransmitter release is mediated by two separate, com- neurotransmitter release, using a previously established lenti- peting pathways: synchronous and asynchronous releases. Both viral system that suppresses ϳ70% of neuronal CaM expression (24). We found that although KD of CaM had no significant * This work was supported, in whole or in part, by a National Institutes of Mental effect on asynchronous release, it surprisingly rescued the loss HealthGrant(toT.C.S.).ThisworkwasalsosupportedbyawardsfromNational Alliance for Research on Schizophrenia and Depression (to Z. P. P.). of synchronous release in Syt1 KO neurons. An unbiased □S The on-line version of this article (available at http://www.jbc.org) contains genome-wide gene expression profiling experiment revealed supplemental Tables S1 and S2 and Fig. S1. that the CaM KD induced a dramatic up-regulation of expres- 1 Both authors contributed equally to this work. 2 To whom correspondence may be addressed: Dept. of Molecular and Cellu- lar Physiology, Stanford University, 1050 Arastradero Rd., Palo Alto, CA 94304-5543. Tel.: 650-721-1421; E-mail: [email protected]. 4 The abbreviations used are: CaM, calmodulin; CaMKII, CaM-dependent 3 To whom correspondence may be addressed: Dept. of Molecular and kinase II; Syt, synaptotagmin; KO, knock-out; Syb, synaptobrevin; DIV, Cellular Physiology, Stanford University, 1050 Arastradero Rd., Palo day(s) in vitro; mIPSC, miniature inhibitory postsynaptic currents; VCP, Alto, CA 94304-5543. Tel.: 650-721-1421; E-mail: [email protected]. vasolin-containing protein. 33930 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 44•OCTOBER 29, 2010 Calmodulin Suppresses Synaptotagmin-2 Expression sion of Syt2 and synaptobrevin-1 (Syb1), which are normally ing CaM 1–3; or viruses with shRNAs and expression of shRNA expressed in the forebrain at low levels but are abundant in silent WT CaM cDNA (supplemental Table S1). Only the gene caudal brain regions (27–29). In addition, the expression of expression levels changed after CaM KD and rescued with WT other caudal synaptic genes was increased, whereas expression CaM in both experiments are included in the list. Full gene of rostral synaptic genes was decreased. Moreover, using expression array data are deposited to the NCBI Gene Expres- molecular replacement experiments, we show that the regula- sion Omnibus. tion of Syt2 expression by CaM requires Ca2ϩ binding to only Quantification of mRNA Level by Quantitative Real Time the C-lobe but not the N-lobe of CaM. Thus, our data show that PCR—The cultured cortical neurons were lysed, and total RNA Ca2ϩ binding to CaM regulates neurotransmitter release not was extracted and purified with a RNAqueous micro kit only in the short term by binding to target proteins (12–14, (Ambion) following the manufacturer’s instructions. The 20–24) but also on a longer time frame by modulating the mRNA level of individual genes was then analyzed by one-step expression of presynaptic proteins such as Syt2, thereby influ- quantitative real time PCR system with pre-made TaqMan encing the properties of neurotransmitter release at a synapse. gene expression assays (Applied Biosystems). Briefly, 30 ng of RNA sample in 1 l of volume was mixed with 10 l of TaqMan EXPERIMENTAL PROCEDURES fast universal PCR master mix (twice), 0.1 l of reverse tran- Neuronal Culture—Mouse cortical culture was made as scriptase (50 units/l), 0.4 l of RNase inhibitor (20 units/l), described elsewhere (6, 24). Briefly, the primary cortical neu- 7.5 lofH2O, and 7 l of TaqMan gene expression assay for rons were isolated from postnatal day 0 pups of Syt1 deficient or the target gene (including the forward and reverse primers and Downloaded from wild-type mice, dissociated by papain digestion, and plated on the TaqMan FAM-MGB probe). The reaction mixture was Matrigel-coated circle glass coverslips. The neurons were cul- loaded onto ABI7900 fast real time PCR machine for 30 min of tured in vitro for 13–16 days in minimal essential medium reverse transcription at 48 °C followed by 40 PCR amplification (Invitrogen) supplemented with B27 (Invitrogen), glucose, cycles consisting of denaturation at 95 °C for 1 s and annealing transferrin, fetal bovine serum, and Ara-C (Sigma). and extension at 60 °C for 20 s. The amplification curve was www.jbc.org ⌬⌬ Lentivirus Packaging and Infection of Neuronal Culture—The collected and analyzed with Ct methods for relative quanti- packaging of lentiviruses and the infection of neurons with len- fication of mRNAs. The amount of mRNA of target genes, nor- tiviruses were described previously (24). Briefly, the lentiviral malized to that of an endogenous control and relative to the Ϫ⌬⌬ at UMDNJ RW JOHNSON, on June 4, 2012 expression vector (control vector L309 or the shRNAs carrying calibrator sample, is calculated by 2 Ct. In the current study, vectors) and three helper plasmids, the pRSV-REV, pMDLg/ GAPDH was used as the endogenous control, and the RNA pRRE, and vesicular stomatitis virus G protein were co-trans- samples derived from neurons infected with control vector fected into HEK 293T cells (ATCC, Manassas, VA) at 6, 2, 2, (L309) were used as calibrators.