Calmodulin Suppresses Synaptotagmin-2 Transcription In

Supplemental Material can be found at: http://www.jbc.org/content/suppl/2010/09/08/M110.150151.DC1.html

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 asynchronous 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. The TaqMan gene expres- and 2 ␮g of DNA/25-cm2 culture area, respectively. The tran- sion assays (Applied Biosystems) used in the current study sient transfections were performed with FuGENE 6 transfec- included: Mm00486655_m1 (CaM 1), Mm00849529_g1 (CaM tion reagent (Roche Applied Science) following the manufac- 2), Mm00482929_m1 (CaM 3), Mm00618457_m1 (Lrrtm3), turer’s instructions. Supernatants with viruses were collected Mm00436864_m1 (Syt2), and mouse GAPD (GAPDH) endog- 48 h after transfection. Cortical neuronal culture was infected enous control. Syt9 quantitative real time PCR PrimeTime at 5 days in vitro (DIV) and used for biochemical or physiolog- assay was designed and custom-made through Integrated DNA ical analysis on 14–16 DIV. All of the steps were performed Technologies. under level II biosafety conditions. Electrophysiology—Electrophysiology was performed as Lentiviral Vector Construction—Lentiviral vectors construc- described previously (6, 24, 30). Briefly, the evoked synaptic tions were described previously (24). Human H1 promoter and responses were triggered by a bipolar electrode (FHC, human U6 promoter were cloned into lentiviral backbone vec- CBAEC75 Concentric Bipolar Electrode OP: 125 ␮m SS; IP: 25 tor FG-12 vector. Cloning sites after H1 promoter are XhoI- ␮m Pt/lr) placed at a position 100–150 ␮m from the soma of XbaI-HpaI; cloning sites after U6 promoter are AscI-ClaI-Rs- neurons recorded. The patch pipettes were pulled from borsili- rII-PacI. Internal ribosome entry site-enhanced GFP was cate glass capillary tubes (Warner Instruments; catalog number cloned in after ubiquitin C promoter, leaving BamHI-EcoRI 64-0793) using PP830 or PC-10 pipette puller (Narishige). The sites for inserting rescue cDNAs. Short hairpin sequences for resistance of pipettes filled with intracellular solution varied CaMs were the same as described previously (24). between 4 and 5 MOhm. After formation of whole cell config- Microarray Expression Assays—The cultured cortical neu- uration and equilibration of intracellular pipette solution, the rons were lysed, and total RNA was extracted and purified with series resistance was adjusted to 8–10 MOhm. Synaptic cur- a RNAqueous micro kit (Ambion) following the manufacturer’s rents were monitored with Multiclamp 700B amplifier (Molec- instructions. Standard gene expression analyses were per- ular Devices). The frequency, duration, and magnitude of extra- formed using the Affymetrix mouse gene ST_1.0 chip by the cellular stimulus were controlled with a model 2100 isolated Protein and Nucleic Acid Facility at Stanford University. Array pulse stimulator (A-M Systems, Inc.) synchronized with data were analyzed using the Partek genomics suite. Gene Clampex 9 data acquisition software (Molecular Devices). The expression levels were compared with their control groups whole cell pipette solution contained 135 mM CsCl, 10 mM individually. Two data sets of CaM KD and CaM KD ϩ WT HEPES, 1 mM EGTA, 1 mM Na-GTP, 4 mM Mg-ATP, and 10 mM CaM rescue were obtained with two batches of cultured neu- QX-314 (pH 7.4, adjusted with CsOH). The bath solution con- rons infected with control viruses; viruses with shRNAs target- tained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2,2mM CaCl2,10

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washed twice using PBS. The materials were directly collected by SDS protein sample buffers (50 ␮lof sample buffer/well for 24-well plates). Equal amounts of samples (25 ␮l) were analyzed by SDS-PAGE and immunoblotting using antibodies as follows: CaM monoclonal antibody (Milllipore; 05-173, 1:1,000); Syt2 (I735, 1:3,000); Syt1 (Cl41.1, 1:4,000); syntaxin 1 (U6251, 1:3,000); SNAP-25 (P913, 1:1,000); Rab3A (42.2, 1:2,000); rabphilin (I731, 1:1,000), secretory carrier membrane proteins (R806, 1:1,000); PSD95 (L667, 1:1,000); Munc18 (J371, 1:1,000); cysteine string protein (R807, 1:1,000); N-ethyl- Downloaded from maleimide-sensitive factor (P944, 1:1,000); NL1 (4C12, 1:1,000); NL2 (169C, 1:200); GDP dissociation inhibitor (81.2, 1:2,000); synaptot-

brevin-1 (P938, 1:500); ␤-actin www.jbc.org (mouse monoclonal antibody clone 14; BD Transduction Labs; 1:2,000); and vasolin-containing protein (VCP; K330, 1:1,000). For protein at UMDNJ RW JOHNSON, on June 4, 2012 quantitations, 125I-labeled secondary antibodies and PhosphoImager detection (Molecular Dynamics) were used. GDP dissociation inhibitor and VCP were employed as internal standards. Data Analysis—The electro- physiological currents were sam- pled at 10 kHz and analyzed off- line using Clampfit 9 (Molecular FIGURE 1. CaM knockdown reduces mini release in Syt1 KO neurons. Cultured cortical neurons from Syt1 KO mice were infected with control lentivirus or with CaM KD lentivirus expressing CaM shRNAs without or with Devices) software. For graphic rep- wild-type CaM rescue mRNA (ϩWT CaM) (24). Neurons were cultured from newborn mice, infected at DIV 5, resentation of the current traces and analyzed at DIV 14–15. A, representative immunoblots of neurons probed with antibodies to CaM, Syt1, shown for evoked synaptic trans- and syntaxin-1 (Synt-1) and visualized by enhanced chemiluminescence. B, representative traces of mIPSCs. C, cumulative distributions of the inter-event intervals of mIPSCs. The plot shows the averages of minis from mission, stimulus artifacts were five neurons. p Ͻ 0.001 for control versus CaM KD; the values for control versus CaM KD with rescue were not removed. For measurements of fre- significant. A Kolmogorov-Smirnov test was used. D, cumulative distributions of the mIPSC amplitudes. The plot shows averages of minis from five neurons. The values for control versus CaM and control versus CaM KD quency of spontaneous release and with rescue were not significant. A Kolmogorov-Smirnov test was used. E, summary graphs of the mIPSC amplitudes of synchronous IPSCs frequency. F, summary graphs of the mIPSC amplitude. G, summary graphs of the 10–90% rise time of mIPSCs. during stimulus trains, individual H, summary graphs of the 90% to 10% decay time of mIPSCs. The data shown are the means Ϯ S.E.; n ϭ number of cells indicated in the bars from three independent cultures. ***, p Ͻ 0.001 as assessed with Student’s t test. mIPSCs or IPSCs were collected using pClamp template search func- mM HEPES, and 10 mM glucose (pH 7.4, adjusted with NaOH). tion. Cumulative distributions of inter-event interval and Inhibitory and excitatory postsynaptic currents were pharma- amplitude of mIPSCs were compared using a Kolmogorov- cologically isolated by adding AMPA and NMDA receptor Smirnov test. All of the statistical comparisons were made using blockers 6-cyano-7-nitroquinoxaline-2,3-dione (20 ␮M) and Student’s t test except where otherwise stated. ␮ ␮ AP-5 (50 M) or GABAA receptor blockers bicuculine (20 M) or picrotoxin (50 ␮M) to the extracellular bath solution. Spon- RESULTS taneous miniature inhibitory postsynaptic currents (mIPSCs) CaM KD Rescues Synchronous Release in Syt1 KO Synapses— were monitored in the presence of tetrodotoxin (1 ␮M) to block We used shRNAs targeting all CaM isoforms (24) to suppress the action potentials. CaM expression in cultured cortical neurons from Syt1 KO Miscellaneous Procedures—For immunoblotting analyses mice. Immunoblotting analysis with enhanced chemilumines- from cultured neurons, at 14–15 DIV, the cultures were cence detection suggested that the CaM KD strongly sup-

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found that instead of impairing asynchronous release, the CaM KD reversed the loss of synchronous release in Syt1 KO neurons (Fig. 2, A–C). Analysis of the kinetics of evoked responses revealed that upon CaM KD, the massively delayed release reaction in Syt1 KO synapses is accelerated to wild-type levels (Fig. 2C). Again, this CaM KD phenotype could be fully rescued by expression of wild-type CaM. Moreover, the restoration of synchronous release is also evident during trains of stimulation (Fig. 2D). However, the presence of synaptic facilitation instead of depression during the stimulus trains indicated Downloaded from that the release probability of CaM KD synapses was lower than that of WT synapses (Fig. 2E), consistent with our previous observation that

the CaM KD decreases the presynap- www.jbc.org tic release probability by a CaMKII- dependent mechanism (24). Gene Expression Profiling Identifies FIGURE 2. CaM KD restores synchronous release in Syt1 KO neurons. Cultured cortical neurons from Syt1 KO and littermate wild-type mice were infected with control lentivirus or with CaM KD lentivirus expressing CaM Multiple Synaptic CaM Targets— at UMDNJ RW JOHNSON, on June 4, 2012 shRNAs without or with wild-type CaM rescue mRNA (24). Neurons were cultured from newborn mice, infected To search for a potential mecha- at DIV 5, and analyzed at DIV 14–15. A, representative traces of evoked IPSCs in Syt1 KO neurons. B, summary graphs of the peak amplitudes of evoked IPSCs (means Ϯ S.E.; numbers of neurons analyzed are shown in bars; nism that accounts for the rescue of n Ͼ 3 independent cultures; ***, p Ͻ 0.001 per Student’s t test). C, time course of the synaptic IPSC charge synchronous release by the CaM KD transfer induced by isolated action potentials. The curves are the averages of n ϭ 11 neurons in each group. in Syt1 KO neurons, we performed D, representative traces of IPSCs evoked by 10 stimuli at 10 Hz in control or CaM KD Syt1 KO neurons and in wild-type neurons. E, normalized synchronous IPSC amplitudes during the 10 Hz stimulus train in CaM KD Syt1 an unbiased gene expression analy- KO (n ϭ 22) and wild-type (n ϭ 10) neurons. The data are the means Ϯ S.E.; the numbers in the bars indicate the sis in cortical neurons. To avoid number of cells analyzed in at least three independent experiments; statistical significance was calculated by artifacts induced by the Syt1 KO or Student’s t test (p Ͻ 0.01). Numerical data are listed in supplemental Table S2. by off-target effects, we used wild- presses CaM expression (Fig. 1A), and quantitations of the CaM type neurons and directly compared neurons that had been mRNA and protein levels confirmed an actual suppression of infected with lentiviruses expressing the CaM shRNAs either CaM expression by ϳ70% (24). without or with a wild-type CaM rescue protein. We then ana- Syt1 functions not only as a Ca2ϩ sensor for synchronous lyzed the gene expression patterns in these neurons with the release but also for spontaneous miniature synaptic release Affymetrix mouse gene ST_1.0 chip. We identified in two inde- (“minis”); it additionally acts as a clamp for mini release (31). As pendent array studies ϳ250 genes whose expression was con- a result, the Syt1 KO causes a large increase in the frequency of sistently up- or down-regulated by the CaM KD, as compared minis; however, the increased minis in Syt1-deficient synapses with the CaM KD/rescue control (see Fig. 4; Table 1 and sup- remain Ca2ϩ-sensitive and are likely mediated by a secondary, plemental Table S1; deposited to the NCBI Gene Expression as yet unidentified Ca2ϩ sensor that exhibits the same proper- Omnibus. ties as the Ca2ϩ sensor for asynchronous release. Thus, we first As expected, multiple classes of genes were regulated by tested whether the CaM KD alters the increased minis observed CaM. Consistent with previous studies (32, 33), we found that in the absence of Syt1. Indeed, we found that the CaM KD activity-dependent genes, such as Homers, Npas2, Arc, and reduced the frequency of mini IPSCs in Syt1 KO neurons ϳ40% Egr3 (supplemental Table S1), were down-regulated by the (Fig. 1, B, C, and E). This reduction in mini release was fully CaM KD. Interestingly, we observed that several synaptic traf- rescued by expression of shRNA-resistant wild-type CaM using ficking proteins were either up- or down-regulated by the CaM the same lentivirus (24), demonstrating that the mini reduction KD (Fig. 3 and supplemental Table S1). Among these was a is not a result of an off-target effect of the shRNAs used for the large increase in the expression of Syt2, which can serve as a experiment. No obvious changes have been observed in the Ca2ϩ sensor for synaptic exocytosis (3, 7–9); thus, this up-reg- amplitudes of mIPSCs after CaM KD (Fig. 1, D and F) and kinet- ulation of Syt2 by the CaM KD likely accounts for the rescue of ics of mIPSCs (Fig. 1, G and H). the Syt1 KO phenotype. In addition, expression of Syb1 was We next examined evoked asynchronous release in Syt1 KO massively increased, whereas expression of Syt4, Syt9, and syn- cultured neurons with or without rescue. To our surprise, we taxin-1A was decreased. Another intriguing class of proteins

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TABLE 1 Correlation of gene expression changes induced by the CaM KD in cortical neurons with the rostral-caudal expression patterns of these genes as deduced from the Allen Brain Atlas Array data fold changes are the averages of two independent experiments. The expression levels at different brain regions were obtained from the Allen Brain Atlas. An up arrow depicts increase; a down arrow indicates decrease; a left arrow indicates that expression levels are higher in rostral than caudal brain regions; and a right arrow indicates the opposite. The ratio of caudal/rostral gene expression was calculated from the data of the Allen Brain Atlas by dividing the numerical values listed there as follows: the average values of medulla and pons: the average values of cortex and hippocampus. Rostral to caudal Expression level -Fold change Ratio Gene name Description expression level in CaM KD (microarray) (CaM KD/control) (caudal/rostral) (Allen Brain Atlas) Syt2 synaptotagmin 2 1 5.64 3 3.04 Cntn2 contactin 2 1 2.22 3 2.02 Lgi3 leucine-rich repeat LGI family, member 3 1 2.12 3 1.12 Coro6 coronin, actin binding protein 6 1 3.96 3 2.06 Flt3 FMS-like tyrosine kinase 3 1 2.56 3 2.79 Phospho1 phosphatase, orphan 1 1 2.15 3 4.11 Aldh1a7 aldehyde dehydrogenase family 1, subfamily A7 1 1.79 3 2.35 Tspan17 tetraspanin 17 1 1.71 3 1.44 Tspan2 tetraspanin 2 1 1.75 3 1.26 F3 coagulation factor III 1 1.46 3 2.90 Stx1a syntaxin 1A 2 0.61 4 0.30 Icam5 intercellular adhesion molecule 5 2 0.54 4 0.02 Lrrtm1 leucine-rich repeat transmembrane neuronal 1 2 0.69 4 0.05 Opcml opioid-binding protein 2 0.6 4 0.37 Downloaded from Lrrtm3 leucine-rich repeat transmembrane neuronal 3 2 0.62 4 0.47 Npas2 Neuronal PAS domain protein 2 2 0.65 4 0.14 NueroD6 neurogenic differentiation 6 2 0.38 4 0.46 Arpc5 actin-related protein 2 2 0.63 4 0.11 Mtap9 microtubule-associated protein 9 2 0.67 4 0.54 Dlg3 discs, large homolog 3 2 0.64 4 0.25 Necab1 N-terminal EF-hand calcium binding protein 1 2 0.61 4 0.27 Doc2B double C2 domain protein B 2 0.58 4 0.18 www.jbc.org Cpne5 copine 5 2 0.56 4 0.23 Sez6 Seizure related gene 6 2 0.63 4 0.34 Ypel2 Yipppee-like 2 2 0.6 4 0.57 Galnt9 UDP-N-acetyl-a-D-galactosamine 2 0.58 4 0.52 2 4 Tiam2 T-cell lymphoma invasion and metastasis 2 0.62 0.14 at UMDNJ RW JOHNSON, on June 4, 2012 Ctxn1 cortexin 1 2 0.6 4 0.20 Pak7 p21 (CDKN1A)-activated kinase 7 2 0.58 4 0.07 Rab40b Rab40b, member RAS oncogene family 2 0.62 4 0.30 Rimbp2 RIMS binding protein 2 2 0.52 4 0.72 Tmem74 transmembrane protein 74 2 0.55 4 0.25 Lingo1 leucine-rich repeat and Ig domain containing 1 2 0.56 4 0.22 Epha6 Eph receptor A6 2 0.56 4 0.62 Ephb6 Eph receptor B6 2 0.69 4 0.45 whose expression was strongly regulated by CaM were cell ciency even under rescue conditions. CaM mRNA levels were adhesion molecules, such as the synaptic cell adhesion mole- low in CaM KD samples and remained low even under rescue cules Lrrtm1, Lrrtm3, and contactin-2 (Fig. 3). Moreover, we conditions (Fig. 4A). Thus, our results indicate that the lentivi- observed up-regulation of sodium channels, and a down-regu- rally mediated KD of CaM is very effective in cultured neurons. lation of potassium channels, suggesting that CaM might con- Next, we analyzed the expression of selected proteins trol the activity-dependent regulation of neuronal excitability. encoded by the mRNAs that were altered by the CaM KD. Finally, we detected changes in multiple genes encoding tran- Immunoblotting confirmed that the CaM KD produced a scription factors, intracellular signal transduction proteins, ele- strong induction of Syt2 and Syb1 protein, consistent with the ments of the cytoskeleton, or metabolic enzymes (supplemental microarray data (Fig. 4B). Syt2 and Syb1 were expressed at very Table S1). It should be noted, however, that despite these mul- low levels in control cortical cultures that only express tifarious changes, more than 95% of genes showed no CaM enhanced GFP; however, in CaM KD condition we found obvi- KD-induced change, suggesting that the observed CaM KD-de- ous expression of both Syt2 and Syb1 (Fig. 4B). Again, the up- pendent expression changes are specific. regulation of Syt2 and Syb1 can be reduced (rescued) by over- Validation of Microarray Results by Quantitative Real Time expression of wild-type CaM in CaM KD neurons. PCR and Immunoblotting—We validated the microarray To achieve a more quantitative understanding of the changes results by quantification of the mRNAs for three representative in protein expression upon CaM KD, we measured the levels of genes. Quantitative real time PCR measurements confirmed 14 synaptic proteins in the CaM knockdown neurons using that Syt2 expression, tested because of its Ca2ϩ sensor function, quantitative immunoblotting with 125I-labeled secondary anti- was up-regulated ϳ10-fold by the CaM KD, whereas Lrrtm3 bodies and PhosphoImager detection. In this analysis, we not and Syt9 expression were down-regulated ϳ2-fold (Fig. 4A). In only analyzed neurons infected with control and CaM KD len- addition, because we are employing a rat CaM2 cDNA to rescue tiviruses but also neurons in which the CaM KD viruses addi- the mouse CaM KD phenotype and because the TaqMan tionally produced shRNA-resistant mRNAs encoding either assays for mouse CaM3 that we employed to measure the wild-type rat CaM or mutant rat CaM that contains mutations 2ϩ mRNA levels do not detect the rat CaM mRNA, we were able to in all four Ca -binding sites (called CaM1,2,3,4). These mea- use quantitative real time PCR to confirm the CaM KD effi- surements further confirmed the array data, demonstrating a

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down-regulated, such as Syt9 and Lrrtms, are primarily expressed in rostral brain regions. Indeed, quantitation of the ratio of expression of selected genes in rostral versus caudal brain regions using the Allen Brain Atlas reveals a tight correlation of the direction of the gene expression change induced by the CaM KD and the rostral-caudal expression gradient of a gene (Table 1). This suggests that although not all genes expressed in a rostral-caudal gradient are subject to CaM regulation in cultured cortical neurons, those whose expression is regulated by CaM in this system are expressed in a predictable gradient in the brain. These observations led us to hypothesize that possibly CaM itself is expressed in a rostral-caudal gradient in brain and may contribute to the specification of rostral-caudal gene expression. We thus measured the levels of CaM, Syt1, Syt2, Syb1, and VCP (as a load control) in the cortex, cerebellum, and spinal

cord of four adult mice by quantitative immunoblotting. Strik- Downloaded from ingly, we found that CaM was expressed in a rostral-caudal gradient, with an absolute level in spinal cord that is nearly 40% lower than in cortex (Fig. 6). Syt1 exhibited an even more pro- nounced rostral-caudal expression gradient, whereas Syt2 and Syb1 displayed an inverse rostral-caudal gradient (Fig. 6). CaM Control of Syt2 Expression Is Independent of CaMKII␣— www.jbc.org The Ca2ϩ dependence of the CaM-mediated suppression of Syt2 expression prompted us to ask whether Syt2 expression in cultured neurons is activity-dependent. However, inhibition of at UMDNJ RW JOHNSON, on June 4, 2012 neuronal activity in cultured cortical neurons using tetrodotoxin (a Naϩ channel inhibitor), AP-5 (an NMDA receptor inhibitor), or nifedipine (an L-type Ca2ϩ channel blocker) had no significant effect on Syt2 expression (supplemental Fig. S1). This result is consistent with the notion that the Ca2ϩ/CaM- dependent control of Syt2 expression does not operate on a short term basis in neurons but is a developmental process. We previously found that in presynaptic terminals, one pathway by which CaM controls synaptic strength is through the activation of CaMK II (24). Because in T lymphocytes, CaM activates CaMKII␣ to inhibit IL2 gene expression (34, 35), we FIGURE 3. Profiling of gene expression in control, CaM KD, or CaM KD with tested whether overexpression of a constitutively active mutant WT CaM rescue. Cultured cortical neurons from newborn wild-type mice of CaMKII␣ (CaMKII␣T286D) reverses the activation of Syt2 were infected at DIV 5 with control lentivirus or with CaM KD lentivirus gene expression upon CaM KD. The rationale for this experi- expressing CaM shRNAs without or with wild-type CaM rescue mRNA and analyzed using Affimetrix arrays at DIV 14–15. A, heat map plot of gene ment is that the same CaMKII␣ mutant rescues the decrease in expression changes in CaM KD neurons without or with wild-type CaM res- synaptic strength induced by CaM KD (24), suggesting that cue, as compared with control neurons. Only genes with CaM KD-induced changes that were rescued by wild-type CaM are shown (n ϭ 2 independent it may represent a general pathway of CaM action. How- experiments). B, functional classification of genes changed by the CaM KD ever, immunoblotting demonstrated that neither wild-type and rescued by wild-type CaM. CaMKII␣ nor constitutively active mutant CaMKII␣T286D reversed the increase in Syt2 expression induced by the CaM large increase in Syt2 levels induced by the CaM knockdown KD (supplemental Fig. S1B), indicating that the suppression of (Ͼ10-fold), and additionally revealed changes in multiple other Syt2 gene expression by CaM is independent of CaMKII␣. ϩ proteins, especially in rabphilin, whose levels decreased Ͼ60% Ca2 Binding to the C-lobe of CaM Sufficed to Suppress Syt2 (Fig. 5). Note that in this analysis the lack of rescue by mutant Expression—CaM suppression of Syt2 expression in cultured 2ϩ 2ϩ CaM1,2,3,4 that is unable to bind Ca provides a further control cortical neurons requires Ca binding, because the CaM ϩ that ensures the specificity of the results. mutant in which all four Ca2 -binding sites were abolished

CaM-regulated Gene Expression Correlates with a Rostral- (CaM1,2,3,4) was unable to rescue the CaM KD phenotype (Fig. Caudal Gradient—Inspection of the gene expression changes 5). The two lobes of CaM, the C- and N-lobes, each contain two induced by CaM suggests that the genes that are up-regulated E-F hand Ca2ϩ-binding sites. Because studies on CaM-regu- by the CaM KD, such as Syt2 and Syb1, are preferentially lated ion channels uncovered a differential requirement for expressed in caudal brain regions, whereas genes that are Ca2ϩ binding to the N- or C-terminal lobes of CaM (36, 37), we

OCTOBER 29, 2010•VOLUME 285•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 33935 Calmodulin Suppresses Synaptotagmin-2 Expression

fully rescued the increased Syt2 expression induced by the CaM KD, whereas mutant CaM lacking Ca2ϩ binding to the C-terminal lobe was inactive (Fig. 7). Thus, CaM normally suppresses Syt2 expression via Ca2ϩ binding to only its C-terminal lobe. DISCUSSION In this study, we tested whether Ca2ϩ binding to CaM triggers asyn- FIGURE 4. Validation of gene expression in CaM KD by quantitative real time PCR and immunoblotting. chronous neurotransmitter release, Cultured cortical neurons from newborn wild-type mice were infected at DIV 5 with control lentivirus or with prompted by the similarities be- CaM KD lentivirus expressing CaM shRNAs without or with wild-type CaM rescue mRNA and analyzed at DIV 14. A, quantitative real time PCR measurements of the mRNA levels of Syt2, Lrrtm3, Syt9, and CaM3. Note that the tween the deduced properties of the ϩ CaM3 mRNA levels are not rescued by expression of the rescue CaM2 cDNA. B, immunoblotting analysis of Syt1, asynchronous release Ca2 sensor Syt2, CaM, and VCP (used as load control). (3) and CaM (15–17) and by previous suggestions that CaM may func- Downloaded from tion as a Ca2ϩ sensor of vesicle exocytosis (25, 26). As an approach, we used cultured cortical neurons from neonatal mice and lentivirally deliv-

ered shRNAs that efficiently sup- www.jbc.org press expression of all CaM isoforms in neurons (Fig. 1A). Our initial experiments focused on neu-

rons from Syt1 KO in which syn- at UMDNJ RW JOHNSON, on June 4, 2012 chronous release is deleted; thus, asynchronous release can be studied in isolation in these neurons, and changes in asynchronous release are easily detected. To our surprise, however, we found that the CaM KD did not decrease asynchronous release measurably but instead partly rescued the loss of asynchronous release in the Syt1 KO neurons (Figs. 1 and 2). Thus, CaM is not the Ca2ϩ sensor for asynchronous release but instead normally suppresses a pathway of synchronous release in Syt1 KO neurons that is redundant with Syt1. In our experimental paradigm investigating cultured neurons from newborn mice, we studied not only FIGURE 5. Quantitation of CaM KD-induced changes in protein levels. Cultured cortical neurons from newborn wild-type mice were infected at DIV 5 with control lentivirus or with CaM KD lentivirus expressing CaM the workings of synapses but also shRNAs without a CaM mRNA or with either wild-type CaM rescue mRNA (ϩ WT CaM) or a mutant CaM rescue the maturation of neurons. In the ϩ mRNA ( CaM1,2,3,4) and analyzed at DIV 14. A, representative immunoblots of neuronal proteins using primary period between the culture and len- antibody against the different proteins as indicated and 125I-conjugated secondary antibodies followed by PhosphoImager detection. B, summary graphs of protein levels measured by quantitative immunoblotting. tiviral infection of the neurons and The data shown are the means Ϯ S.E. *, p Ͻ 0.05; **, p Ͻ 0.01 as analyzed by Student’s t test (n ϭ 3 independent their analysis 2 weeks later, the neu- cultures). Synt, syntaxin-1; S-25, SNAP-25; CSP, cysteine string protein; NL1, neuroligin-1; M18, Munc18-1; Rph, rabphilin; SC., secretory carrier membrane protein; GDI, GDP dissociation inhibitor; NL2, neuroligin-2. rons developed from immature cells to morphologically and functionally advanced neurons; at the time of asked whether all CaM Ca2ϩ-binding sites are required for sup- plating, the neurons lacked dendrites, axons, spines, and syn- pression of Syt2 expression or whether Ca2ϩ binding to one of apses, whereas at the time of analysis, all of these had developed. the two lobes is sufficient. Strikingly, mutant CaM in which Thus, we hypothesized that the CaM KD may have activated Ca2ϩ binding to the N-terminal lobe of CaM was blocked still synchronous release in Syt1 KO neurons by inducing changes

33936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 44•OCTOBER 29, 2010 Calmodulin Suppresses Synaptotagmin-2 Expression Downloaded from www.jbc.org

FIGURE 6. Quantitative analysis of CaM, Syt1, Syt2, and Syb1 levels in cortex, cerebellum, and spinal cord of mice. A, representative immunoblots of mouse cortex, cerebellum/brain stem, and spinal cord homogenates using primary antibody against Syt1, Syt2, Syb1, and CaM and 125I-conju- at UMDNJ RW JOHNSON, on June 4, 2012 gated secondary antibodies. B, quantitation of Syt1, Syt2, Syb1, and CaM expression levels normalized to VCP in different brain regions. VCP was used as internal loading control because it is ubiquitously expressed without nota- ble differences between cell types. The data shown are the means Ϯ S.E. **, p Ͻ 0.01; ***, p Ͻ 0.001 as analyzed by Student’s t test (n ϭ 4 mice). in gene expression. To explore this hypothesis, we performed whole genome array studies that led to the identification of a cohort of genes that were up- or down-regulated by the CaM KD (Fig. 3). Strikingly, Syt2 was among the up-regulated genes, and its activation by the CaM KD was confirmed by quantitative mRNA and protein level measurements (Figs. 4 and 5). Because 2ϩ Syt2 normally acts as a fast Ca sensor for neurotransmitter ؉ FIGURE 7. Ca2 binding to the C-lobe of CaM regulates Syt2 expression in release in hindbrain regions, such as the brain stem (3, 10), the cortical neurons. Cultured cortical neurons from newborn wild-type or Syt1 increase in Syt2 expression upon CaM KD provides a facile KO mice were infected at DIV 5 with control lentivirus, with CaM KD lentivirus expressing CaM shRNAs without a CaM rescue construct, or with either wild- explanation for the reversal of the Syt1 KO phenotype by the type CaM (ϩ WT CaM) or mutant CaM in which Ca2ϩ binding to the N-lobe CaM KD. Note that in our experiments, all of the shRNA-de- (CaM1,2), the C-lobe (CaM3,4), or both lobes of CaM (CaM1,2,3,4) is blocked. The pendent effects are controlled for by rescue experiments, an neurons were analyzed at DIV 14–15. A, schematic structures of wild-type and mutant CaMs. B, immunoblot analysis of the indicated proteins in control and essential component given the many off-target effects of CaM KD neurons expressing various CaM constructs. Note that to better indi- shRNAs. cate the expression level of Syt2, we did two different exposure times for the immunoblots. The short exposure (short exp.) was ϳ15 s, and the long expo- The gene expression changes induced by the CaM KD were sure (long exp.) was ϳ5 min. C, representative traces of IPSCs monitored in relatively restricted, affecting only ϳ250 genes (Fig. 3 and sup- Syt1 KO neurons that were infected with the indicated lentiviruses. D, sum- plemental Table S1). Analysis of these gene expression changes mary graphs of the peak amplitudes of evoked IPSCs. The data shown are the means Ϯ S.E. ***, p Ͻ 0.001 as analyzed by Student’s t test (n ϭ number of yielded several observations. First, the expression of multiple neurons indicated in bars from three independent cultures. E, kinetic analysis genes involved in neurotransmitter release was affected (e.g. of the IPSC time course (n ϭ 10–17 in each group). Syb1, syntaxin-1A, and Syt9 in addition to Syt2); this suggests that the multifarious functions of CaM at the synapse, functions ing and the cytoskeleton, the CaM KD also specifically altered that go beyond simply regulating ion channels, signal transduc- the expression of cell adhesion molecules. We found that cell tion, and Munc13, include regulating synaptic gene expression adhesion molecules such as cntnap1 (contactin associate pro- during development. Second, apart from the expected changes tein 1, CASPR) were up-regulated, whereas other cell adhesion in the expression of activity-dependent genes (e.g. Egr3, Npas2, molecules such as Lrrtms and latrophilins/CLs were down-reg- Arc, and Homer) and of genes involved in intracellular signal- ulated by the CaM KD. Lrrtms have been shown to be the

OCTOBER 29, 2010•VOLUME 285•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 33937 Calmodulin Suppresses Synaptotagmin-2 Expression

dal expression pattern of the genes involved. Thus, CaM mediates a Ca2ϩ-dependent regulation of synaptic transmission that goes beyond its acute role in pre- and postsynaptic compart- ments toward specifying the composition of synapses.

Acknowledgments—We thank Dr. John Adelman (Vollum Institute) for CaM Ca2ϩ-binding mutant constructs and Kaishan Xian and Iryna Huryeva for excellent technical assistance.

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OCTOBER 29, 2010•VOLUME 285•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 33939

SUPPLEMENTARY MATERIALS for “Calmodulin suppresses synaptotagmin-2 transcription in cortical neurons” by Zhiping P. Pang1,3*, Wei Xu1,2*, Peng Cao2, and Thomas C. Südhof1,2, 3

Supplementary Figure 1 Activity blockade and activation of CaM Kinase IIα do not increase the expression of Syt2. A. Activity blockade does not activate Syt2 expression in cultured cortical neurons from wild-type mice. Neurons were treated at DIV12 with TTX (1 µM, to block action potentials), DL-APV (50 µM, to block NMDA receptors) or nifedipine (20 µM, to block L-type Ca2+-channels), and harvested after 48, 72, or 96 hrs. To ensure the continued activity of different blockers, the same amounts of blockers were added again at 48 hrs for the 72 and 96 hrs treatments. Samples were analyzed by immunoblotting for the indicated proteins Downloaded from (abbreviations same as above). B. CaMKIIα does not mediate the CaM-dependent suppression of Syt2 expression. Cultured cortical neurons from newborn wild-type mice were infected at DIV5 with control lentivirus or with CaM KD lentivirus expressing CaM shRNAs without a rescue construct, or with wild-type CaMKIIα or constitutively active mutant CamKIIαT286D as rescue constructs. Neurons were analyzed by immunoblotting for Syt2 and www.jbc.org syntaxin-1 (Synt.) at DIV 14.

at UMDNJ RW JOHNSON, on June 4, 2012

Supplemental Table 1 List of genes whose expression levels are affected by CaM-RNAi

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

Category 1: Transcription Regulation

NM_001081338 L3mbtl l(3)mbt-like (Drosophila) 1.59 0.71 1.77 1.12 scratch homolog 1, zinc finger NM_130893 Scrt1 protein (Drosophila) 1.51 0.93 1.42 0.97 nucleosome assembly protein 1- NM_008671 Nap1l2 like 2 1.49 1.06 1.46 0.97 recombination signal binding NM_009035 Rbpj protein for immunoglobulin kap 1.48 1.20 1.79 1.11

NM_001077398 Ldb2 LIM domain binding 2 0.71 0.87 0.66 1.31

NM_019743 Rybp RING1 and YY1 binding protein 0.69 1.05 0.49 1.03

NM_173780 Klf8 Kruppel-like factor 8 0.69 1.08 0.60 1.06

NM_053123 Smarca1 SWI 0.67 1.03 0.50 0.93

NM_025282 Mef2c myocyte enhancer factor 2C 0.65 0.89 0.54 1.23

NM_009769 Klf5 Kruppel-like factor 5 0.65 1.03 0.66 0.99

NM_008719 Npas2 neuronal PAS domain protein 2 0.65 1.20 0.64 1.07

NM_009234 Sox11 SRY-box containing gene 11 0.62 1.00 0.61 1.06

NM_175045 Bcor Bcl6 interacting corepressor 0.60 1.10 0.67 1.34

NM_024124 Hdac9 histone deacetylase 9 0.58 1.07 0.65 1.20

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

NM_009717 Neurod6 neurogenic differentiation 6 0.57 1.87 0.18 0.88 calmodulin binding transcription NM_001081557 Camta1 activator 1 0.53 0.90 0.63 1.19

NM_018781 Egr3 early growth response 3 0.30 1.18 0.59 1.53

Category 2: Membrane Receptors solute carrier family 2 (facilitated NM_172659 Slc2a6 glucose transporter) 2.12 0.88 2.09 0.99 solute carrier family 13 (sodium- NM_054055 Slc13a3 dependent dicarboxylate 1.69 0.82 1.65 0.77 ATP-binding cassette, sub-family A NM_153145 Abca8a (ABC1), member 8a 1.67 0.68 1.40 0.87

NM_178405 Atp1a2 ATPase, Na+ 1.43 1.00 1.41 0.91

NM_030682 Tlr1 toll-like receptor 1 1.42 0.96 1.50 1.21 Downloaded from

NM_009199 Slc1a1 solute carrier family 1 (neuronal) 0.70 1.11 0.63 1.01 protein tyrosine phosphatase, NM_011216 Ptpro receptor type, O 0.70 1.01 0.57 1.15 protein tyrosine phosphatase, NM_008983 Ptprk receptor type, K 0.69 0.88 0.45 1.46 solute carrier family 16 www.jbc.org NM_011391 Slc16a7 (monocarboxylic acid transporter 0.69 0.96 0.70 1.28

NM_177328 Grm7 glutamate receptor, metabotropic 7 0.67 1.20 0.50 1.25

NM_001004761 Gpr158 G protein-coupled receptor 158 0.67 1.06 0.60 1.36 at UMDNJ RW JOHNSON, on June 4, 2012 neurotrophic tyrosine kinase, NM_008746 Ntrk3 receptor, type 3 0.65 1.17 0.55 1.11

NM_010077 Drd2 dopamine receptor 2 0.64 0.87 0.62 1.10

NM_148946 Slc8a2 solute carrier family 8 0.63 1.01 0.54 1.67

NM_145066 Gpr85 G protein-coupled receptor 85 0.61 0.91 0.46 1.11

NM_199058 Gpr6 G protein-coupled receptor 6 0.61 0.87 0.64 1.80

NM_173410 Gpr26 G protein-coupled receptor 26 0.36 1.17 0.46 1.31

Category 3: Membrane Ion Channels

NM_001013390 Scn4b sodium channel, type IV, beta 2.31 1.09 6.81 1.06 calcium channel, voltage- NM_009783 Cacna1g dependent, T type, alpha 1G 1.59 0.70 1.57 1.18 sodium channel, voltage-gated, NM_011322 Scn1b type I, beta 1.47 1.00 1.69 0.88 potassium voltage-gated channel, NM_133207 Kcnh7 subfamily H (eag-related) 0.71 1.14 0.54 1.36 sodium channel, voltage-gated, NM_001099298 Scn2a1 type II, alpha 1 0.71 1.03 0.60 1.30 calcium channel, voltage- NM_009782 Cacna1e dependent, R type, alpha 1E sub 0.68 0.93 0.61 1.25 potassium voltage-gated channel, NM_019697 Kcnd2 Shal-related family, member 2 0.66 0.94 0.54 0.95 glutamate receptor, ionotropic, NM_008170 Grin2a NMDA2A (epsilon 1) 0.65 1.01 0.45 1.00 amiloride-sensitive cation channel NM_001034013 Accn1 1, neuronal 0.65 0.84 0.65 1.13 potassium voltage-gated channel, NM_153512 Kcng3 subfamily G, member 3 0.65 1.07 0.45 1.22 calcium channel, voltage- NM_007583 Cacng2 dependent, gamma subunit 2 0.61 0.95 0.69 0.98 potassium voltage-gated channel, NM_023872 Kcnq5 subfamily Q, member 5 0.60 1.02 0.42 0.99 S2

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res. potassium voltage-gated channel, NM_010601 Kcnh3 subfamily H (eag-related) 0.58 1.10 0.50 1.52 calcium channel, voltage- NM_001044308 Cacna1i dependent, alpha 1I subunit 0.46 0.99 0.47 1.04 calcium channel, voltage- NM_019430 Cacng3 dependent, gamma subunit 3 0.44 1.28 0.64 1.74 potassium channel, subfamily V, NM_026200 Kcnv1 member 1 0.37 1.19 0.47 2.27

Category 4: Cytoskeleton

NM_139128 Coro6 coronin, actin binding protein 6 3.46 0.99 4.45 1.11

NM_010904 Nefh neurofilament, heavy polypeptide 2.78 0.74 2.73 0.62

NM_032393 Mtap1a microtubule-associated protein 1 A 2.42 0.92 2.40 0.87

NM_001039546 Myo6 myosin VI 1.43 0.96 1.43 1.05 Downloaded from

NM_026369 Arpc5 actin related protein 2 0.71 1.07 0.55 0.96

NM_001081230 Mtap9 microtubule-associated protein 9 0.71 0.92 0.63 1.00 discs, large homolog 3 NM_016747 Dlg3 (Drosophila) 0.68 1.00 0.60 0.90 www.jbc.org NM_011983 Homer2 homer homolog 2 (Drosophila) 0.67 1.00 0.52 1.14

NM_019824 Arpc3 actin related protein 2 0.67 1.11 0.61 0.93 discs, large (Drosophila) homolog-

NM_198618 Dlgap3 associated protein 3 0.67 1.20 0.69 1.17 at UMDNJ RW JOHNSON, on June 4, 2012 discs, large (Drosophila) homolog- NM_172910 Dlgap2 associated protein 2 0.66 0.92 0.58 0.93

NM_007864 Dlg4 PSD-95 0.62 0.94 0.69 1.11

NM_021883 Tmod1 tropomodulin 1 0.60 1.21 0.70 1.57 membrane protein, palmitoylated 2 NM_016695 Mpp2 (MAGUK p55 subfamily memb 0.58 0.91 0.54 1.10

NM_021287 Spnb3 spectrin beta 3 0.52 0.90 0.58 1.23

NM_147176 Homer1 homer homolog 1 (Drosophila) 0.45 1.24 0.48 1.20 GRP1 (general receptor for NM_019518 Grasp phosphoinositides 1)-associated 0.36 1.34 0.69 1.49 activity regulated cytoskeletal- NM_018790 Arc associated protein 0.35 1.99 0.71 1.90

Category 5: Calcium signaling (Channels not included)

NM_011242 Rasgrp2 RAS, guanyl releasing protein 2 2.87 1.17 2.03 1.03

NM_011311 S100a4 S100 calcium binding protein A4 1.54 1.09 1.60 0.87 N-terminal EF-hand calcium NM_178617 Necab1 binding protein 1 0.71 1.06 0.51 1.58

NM_134094 Ncald neurocalcin delta 0.69 0.89 0.63 1.02

NM_007873 Doc2b double C2, beta 0.68 0.84 0.48 1.07

NM_009790 Calm1 calmodulin 1 0.60 0.35 0.38 0.26

NM_153166 Cpne5 copine V 0.58 1.38 0.54 1.52

NM_007589 Calm2 calmodulin 2 0.47 0.53 0.34 0.39

NM_010471 Hpca hippocalcin 0.40 0.96 0.40 1.31

NM_007590 Calm3 calmodulin 3 0.40 0.19 0.19 0.09

Category 6: Intracellular signal transduction S3

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

NM_010229 Flt3 FMS-like tyrosine kinase 3 2.32 1.00 2.80 1.06 apoptosis-inducing factor, NM_175178 Aifm3 mitochondrion-associated 3 1.72 0.72 2.51 0.96

NM_010404 Hap1 huntingtin-associated protein 1 1.69 0.79 1.55 0.53

NM_011756 Zfp36 zinc finger protein 36 1.65 1.10 1.61 0.98

NM_008832 Phka1 phosphorylase kinase alpha 1 1.58 0.97 1.54 1.11

NM_008817 Peg3 paternally expressed 3 1.57 0.73 2.15 1.10

NM_013834 Sfrp1 secreted frizzled-related protein 1 1.56 0.96 1.83 1.09 LIM-domain containing, protein NM_010717 Limk1 kinase 1.54 0.90 1.57 0.67

ENSMUST00000055576 Pcsk6 proprotein convertase subtilisin 1.49 0.86 1.53 0.82 Downloaded from

NM_172778 Maob monoamine oxidase B 1.48 0.93 1.76 1.02 recombination signal binding NM_009035 Rbpj protein for immunoglobulin kap 1.48 1.20 1.79 1.11 phosphatidylinositol 3-kinase, NM_029094 Pik3cb catalytic, beta polypeptid 1.46 0.72 2.07 1.04 www.jbc.org NM_172263 Pde8b phosphodiesterase 8B 1.46 0.95 1.44 1.17

NM_007928 Mark2 MAP 0.71 1.01 0.71 0.97

ENSMUST00000076810 Kalrn kalirin, RhoGEF kinase 0.71 0.95 0.59 1.28 at UMDNJ RW JOHNSON, on June 4, 2012 NM_029933 Bcl9 B-cell CLL 0.71 0.85 0.62 0.88 cyclic AMP-regulated NM_033264 Arpp21 phosphoprotein, 21 0.71 0.95 0.62 1.48

NM_207223 Centb5 centaurin, beta 5 0.71 0.98 0.67 0.93

NM_178681 Dgkb diacylglycerol kinase, beta 0.69 1.16 0.70 1.12

NM_153171 Rgs13 regulator of G-protein signaling 13 0.69 1.62 0.67 1.72

NM_011104 Prkce protein kinase C, epsilon 0.69 0.86 0.64 1.16

NM_008083 Gap43 growth associated protein 43 0.69 1.25 0.69 0.98 Rab40b, member RAS oncogene NM_139147 Rab40b family 0.67 1.05 0.57 1.01 connector enhancer of kinase NM_177751 Cnksr2 suppressor of Ras 2 0.66 0.99 0.48 1.24

NM_183315 Ctxn1 cortexin 1 0.65 0.96 0.55 1.01

NM_007634 Ccnf cyclin F 0.65 1.21 0.61 0.86

NM_172858 Pak7 p21 (CDKN1A)-activated kinase 7 0.65 0.90 0.51 1.07

BC024265 Mast3 microtubule associated serine 0.65 1.03 0.68 1.08 SH3-domain kinase binding protein NM_021389 Sh3kbp1 1 0.64 1.24 0.62 0.94

NM_198114 Dagla diacylglycerol lipase, alpha 0.64 0.93 0.61 1.04 F-box and WD-40 domain protein NM_080428 Fbxw7 7, archipelago homolog (Dro 0.64 0.98 0.59 1.01

NM_029761 Dok5 docking protein 5 0.63 1.24 0.67 1.25 protein tyrosine phosphatase, non- NM_011201 Ptpn1 receptor type 1 0.63 0.92 0.65 1.27 T-cell lymphoma invasion and NM_001122998 Tiam2 metastasis 2 0.63 1.02 0.61 1.18

NM_026878 Rasl11b RAS-like, family 11, member B 0.62 1.11 0.70 0.87 S4

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res. Rap guanine nucleotide exchange NM_175930 Rapgef5 factor (GEF) 5 0.60 1.23 0.41 1.19

NM_198419 Phactr1 phosphatase and actin regulator 1 0.60 0.92 0.69 1.17 nuclear receptor subfamily 4, NM_015743 Nr4a3 group A, member 3 0.58 1.17 0.68 1.10 brain-specific angiogenesis NM_130862 Baiap2 inhibitor 1-associated protei 0.56 0.85 0.60 1.20 protein kinase, cGMP-dependent, NM_008926 Prkg2 type II 0.48 1.35 0.63 1.35 wingless-related MMTV integration NM_009528 Wnt7b site 7B 0.47 0.86 0.50 1.04

Category 7 :Vesicle and trafficking

NM_009307 Syt2 synaptotagmin II 5.31 0.84 5.95 0.89

vesicle-associated membrane Downloaded from NM_001080557 Vamp1 protein 1 2.61 1.05 3.06 1.03

NM_016900 Cav2 caveolin 2 1.85 1.15 1.70 1.09

NM_016908 Syt9 synaptotagmin IX 0.69 1.05 0.58 0.99

NM_016801 Stx1a syntaxin 1A (brain) 0.68 1.01 0.54 0.91 www.jbc.org NM_182993 Slc17a7 vGluT1 0.67 0.96 0.49 0.89

NM_009308 Syt4 synaptotagmin IV 0.65 1.09 0.64 0.86

Category 8: Cell Adhesion molecules at UMDNJ RW JOHNSON, on June 4, 2012

NM_177129 Cntn2 contactin 2 2.54 1.06 1.89 0.80

NM_016782 Cntnap1 contactin associated protein-like 1 2.31 0.77 2.19 0.91 leucine-rich repeat LGI family, NM_145219 Lgi3 member 3 1.87 0.63 2.36 0.77 leucine rich repeat transmembrane NM_028880 Lrrtm1 neuronal 1 0.71 0.96 0.66 0.98 FAT tumor suppressor homolog 3 NM_001080814 Fat3 (Drosophila) 0.70 1.19 0.58 1.35

NM_080285 Cttnbp2 cortactin binding protein 2 0.66 0.89 0.66 1.44 leucine-rich repeat LGI family, NM_020278 Lgi1 member 1 0.62 1.11 0.36 1.03 intercellular adhesion molecule 5, NM_008319 Icam5 telencephalin 0.62 0.92 0.45 1.05

NM_177906 Opcml opioid binding protein 0.62 0.91 0.58 0.98

NM_021424 Pvrl1 poliovirus receptor-related 1 0.62 1.12 0.57 1.13

NM_011858 Odz4 odd Oz 0.61 0.93 0.66 1.08

NM_011855 Odz1 odd Oz 0.61 1.01 0.67 1.09 leucine rich repeat transmembrane NM_178678 Lrrtm3 neuronal 3 0.57 1.04 0.67 1.31

NM_001081298 Lphn2 latrophilin 2 0.57 0.84 0.59 1.36

Category 9: Intercellular Signaling R-spondin 2 homolog (Xenopus NM_172815 Rspo2 laevis) 0.71 1.30 0.68 1.06

NM_009472 Unc5c unc-5 homolog C (C. elegans) 0.71 0.98 0.43 0.86

NM_008882 Plxna2 plexin A2 0.71 1.02 0.58 1.06 transmembrane protein with EGF- NM_021436 Tmeff1 like and two follistatin-l 0.71 0.99 0.69 1.11

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

NM_010141 Epha7 Eph receptor A7 0.69 0.94 0.54 0.93

NM_198962 Hcrtr2 hypocretin (orexin) receptor 2 0.69 0.87 0.61 1.07

NM_007938 Epha6 Eph receptor A6 0.68 0.92 0.43 0.84 sema domain, immunoglobulin NM_001126047 Sema4c domain (Ig), transmembrane 0.68 1.13 0.67 1.17

NM_007680 Ephb6 Eph receptor B6 0.66 1.00 0.71 1.10 leucine rich repeat and Ig domain NM_181074 Lingo1 containing 1 0.63 1.28 0.48 1.15 sema domain, immunoglobulin NM_013658 Sema4a domain (Ig), transmembrane do 0.59 1.54 0.50 1.62

NM_009217 Sstr2 somatostatin receptor 2 0.54 1.12 0.52 1.36

NM_031161 Cck cholecystokinin 0.50 1.22 0.35 1.18 Downloaded from sema domain, immunoglobulin NM_009152 Sema3a domain (Ig), short basic doma 0.50 1.85 0.60 1.14

NM_010140 Epha3 Eph receptor A3 0.50 1.40 0.33 1.13

Category 10: Other www.jbc.org NM_030700 Maged2 melanoma antigen, family D, 2 2.68 1.08 2.70 0.80

NM_001040611 Peg10 paternally expressed 10 2.44 0.67 6.11 0.91

NM_027100 Rwdd2a RWD domain containing 2A 2.22 0.86 1.94 0.93 at UMDNJ RW JOHNSON, on June 4, 2012 NM_153104 Phospho1 phosphatase, orphan 1 2.08 1.20 2.22 1.19

NM_153169 Pnma3 paraneoplastic antigen MA3 1.98 0.79 1.80 0.82

NM_009801 Car2 carbonic anhydrase 2 1.74 1.12 1.44 0.96 aldehyde dehydrogenase family 1, NM_011921 Aldh1a7 subfamily A7 1.73 0.99 1.84 1.19

NM_027533 Tspan2 tetraspanin 2 1.65 1.04 1.85 1.09

NM_028841 Tspan17 tetraspanin 17 1.64 1.14 1.78 0.78 complement component 4B (Childo NM_009780 C4b blood group) 1.63 1.11 2.12 0.81

NM_011123 Plp1 proteolipid protein (myelin) 1 1.63 1.07 1.61 1.02

NM_025943 Dzip1 DAZ interacting protein 1 1.57 1.05 1.49 0.94

NM_182991 Tmem59l transmembrane protein 59-like 1.53 0.86 1.67 0.94

NM_001001985 Nat8l N-acetyltransferase 8-like 1.50 1.00 1.42 0.89

NM_010171 F3 coagulation factor III 1.48 1.03 1.43 1.03

NM_013813 Epb4.1l3 erythrocyte protein band 4.1-like 3 1.48 0.78 1.59 0.82 scavenger receptor class A, NM_172604 Scara3 member 3 1.46 1.11 1.82 1.17 fatty acid binding protein 5, ENSMUST00000029046 Fabp5 epidermal 1.46 1.00 1.40 0.96

NM_001038699 Fn3k fructosamine 3 kinase 1.45 0.97 1.55 1.22

ENSMUST00000067439 Prune2 prune homolog 2 (Drosophila) 1.45 0.71 1.82 1.20

NM_009155 Sepp1 selenoprotein P, plasma, 1 1.45 0.87 1.66 1.10

NM_011020 Hspa4l heat shock protein 4 like 1.45 1.07 1.57 1.11 membrane bound C2 domain NM_011843 Mbc2 containing protein 1.44 0.96 2.69 1.06

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

NM_145570 Tmem166 transmembrane protein 166 1.44 0.77 1.57 1.03 ATP-binding cassette, sub-family D NM_011994 Abcd2 (ALD), member 2 1.43 0.86 1.43 0.83

NM_184109 Rtl1 retrotransposon-like 1 1.42 1.00 1.52 1.01

NM_026203 Ahi1 Abelson helper integration site 1 1.41 1.06 1.72 1.07

NM_011157 Srgn serglycin 1.40 0.80 1.63 1.08 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- NM_173739 Galntl4 acetylg 0.70 1.02 0.65 1.07

NM_026898 Wdr53 WD repeat domain 53 0.70 0.87 0.60 0.96

NM_001081146 Prickle2 prickle-like 2 (Drosophila) 0.70 0.96 0.52 1.08

hyaluronan and proteoglycan link Downloaded from NM_013500 Hapln1 protein 1 0.70 1.13 0.60 0.92

NM_173777 Olfm2 olfactomedin 2 0.69 0.90 0.71 0.99

NM_021286 Sez6 seizure related gene 6 0.69 0.98 0.57 1.14

NM_010266 Gda guanine deaminase 0.69 0.88 0.67 1.39 www.jbc.org zinc finger, SWIM domain BC021311 Zswim6 containing 6 0.69 1.06 0.69 0.98

NM_133706 Tmem97 transmembrane protein 97 0.69 1.59 0.67 1.47 at UMDNJ RW JOHNSON, on June 4, 2012 NM_172637 Hectd2 HECT domain containing 2 0.68 1.60 0.60 1.30

NM_001024928 Zfp667 zinc finger protein 667 0.68 1.07 0.63 1.52

NM_009262 Spock1 sparc 0.68 0.93 0.48 1.07 zinc finger, DHHC domain NM_146073 Zdhhc14 containing 14 0.68 0.91 0.48 1.07

NM_009270 Sqle squalene epoxidase 0.68 1.18 0.46 1.20

NM_133706 Tmem97 transmembrane protein 97 0.67 1.27 0.71 1.18

NM_011119 Pa2g4 proliferation-associated 2G4 0.67 1.01 0.67 0.88 cysteine-serine-rich nuclear protein NM_153407 Csnrp2 2 0.67 0.99 0.64 1.11 V-set and transmembrane domain NM_198627 Vstm2l containing 2-like 0.67 1.14 0.57 1.02

NM_011846 Mmp17 matrix metallopeptidase 17 0.67 1.15 0.57 1.04

NM_178920 Mal2 mal, T-cell differentiation protein 2 0.66 1.15 0.65 0.91 U7 snRNP-specific Sm-like protein NM_028185 Lsm11 LSM11 0.66 1.09 0.65 1.08

NM_153155 C1ql3 C1q-like 3 0.66 1.28 0.57 1.05

U88401 Mtag2 metastasis associated gene 2 0.66 0.95 0.71 1.00

NM_199024 Nol4 nucleolar protein 4 0.65 0.84 0.61 1.26 cat eye syndrome chromosome NM_033567 Cecr6 region, candidate 6 homolog (h 0.64 1.05 0.64 1.05

NM_178252 Snx26 sorting nexin 26 0.64 1.03 0.48 0.88

NM_201529 Lmo7 LIM domain only 7 0.64 0.92 0.45 1.12

NM_001033212 Rprml reprimo-like 0.64 1.32 0.65 1.19

NM_175645 Xylt1 xylosyltransferase 1 0.63 0.84 0.59 1.21

NM_001081388 Rimbp2 RIMS binding protein 2 0.62 0.84 0.42 0.95 S7

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

ENSMUST00000074081 Csmd2 CUB and Sushi multiple domains 2 0.62 0.91 0.56 1.18

NM_172434 Tnrc4 trinucleotide repeat containing 4 0.62 0.84 0.42 1.15

NM_001005341 Ypel2 yippee-like 2 (Drosophila) 0.60 1.04 0.60 1.33

NM_175502 Tmem74 transmembrane protein 74 0.60 0.92 0.50 0.96 phytanoyl-CoA hydroxylase NM_145981 Phyhip interacting protein 0.59 0.87 0.41 0.98 phosphodiesterase 4D interacting NM_001039376 Pde4dip protein (myomegalin) 0.58 0.93 0.70 1.24

NM_001081064 Pdzd2 PDZ domain containing 2 0.56 1.10 0.62 1.21

AK032191 Nol4 nucleolar protein 4 0.55 0.98 0.64 1.06 3-hydroxy-3-methylglutaryl- NM_008255 Hmgcr Coenzyme A reductase 0.54 1.41 0.52 1.27 Downloaded from membrane-associated ring finger AK162044 March11 (C3HC4) 11 0.54 1.66 0.70 1.61 deleted in bladder cancer 1 NM_019967 Dbc1 (human) 0.54 1.31 0.68 1.41

NM_019675 Stmn4 stathmin-like 4 0.52 1.00 0.50 0.86 protein arginine N- www.jbc.org NM_201371 Prmt8 methyltransferase 8 0.52 0.90 0.49 0.92 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N-

NM_198306 Galnt9 acetylga 0.46 1.37 0.70 1.03 at UMDNJ RW JOHNSON, on June 4, 2012

Category 11: Uncharacterized

BC052055 BC052055 cDNA sequence BC052055 3.33 0.83 2.66 0.99

BC125016 EG328644 predicted gene, EG328644 2.44 0.67 3.01 1.10

AF529169 AF529169 cDNA sequence AF529169 1.85 1.04 1.99 0.88

NM_001114174 Gm967 gene model 967, (NCBI) 1.60 0.76 1.57 1.05

NM_001081029 4930420K17Rik RIKEN cDNA 4930420K17 gene 1.57 1.04 1.56 1.20

ENSMUST00000049544 2610301F02Rik RIKEN cDNA 2610301F02 gene 1.43 1.14 1.47 1.12

BC055818 D330028D13Rik RIKEN cDNA D330028D13 gene 1.41 1.01 1.56 0.85 DNA segment, Chr 11, Brigham & NM_001039167 D11Bwg0517e Women's Genetics 0 0.71 0.92 0.58 1.27

NM_026279 2310026E23Rik RIKEN cDNA 2310026E23 gene 0.69 1.04 0.69 0.90

BC119515 1700001L19Rik RIKEN cDNA 1700001L19 gene 0.69 0.94 0.57 1.14

NM_144935 BC018242 cDNA sequence BC018242 0.68 0.90 0.52 0.93

NM_001033166 2700094K13Rik RIKEN cDNA 2700094K13 gene 0.67 0.93 0.70 1.02

ENSMUST00000059500 BC028663 cDNA sequence BC028663 0.65 1.04 0.68 0.87

ENSMUST00000057768 4930429B21Rik RIKEN cDNA 4930429B21 gene 0.61 1.23 0.69 1.40 predicted gene, ENSMUST00000061282 OTTMUSG00000013918 OTTMUSG00000013918 0.60 1.00 0.41 1.83

BC072639 2010300C02Rik RIKEN cDNA 2010300C02 gene 0.59 0.90 0.58 1.22

NM_001033391 A130090K04Rik RIKEN cDNA A130090K04 gene 0.57 1.09 0.48 1.41

BC092532 2900046G09Rik RIKEN cDNA 2900046G09 gene 0.51 0.97 0.48 1.06

ENSMUST00000068927 A330084C13Rik RIKEN cDNA A330084C13 gene 0.50 1.13 0.58 0.87

Accession No. Gene Symbol Description Relative mRNA Level CaM KD1 CaM KD2  + Res.  + Res.

XM_001473525 LOC100039795 similar to C1orf32 putative protein 0.46 0.97 0.62 1.32

NM_029530 6330527O06Rik RIKEN cDNA 6330527O06 gene 0.44 1.56 0.69 1.53

ENSMUST00000101196 3110047P20Rik RIKEN cDNA 3110047P20 gene 0.40 0.99 0.29 1.12

NM_182808 C630007B19Rik RIKEN cDNA C630007B19 gene 0.39 1.60 0.28 1.49 Data shown are from two independent experiments performed with neurons that contained the CaM KD shRNAs, wihout or with full-length CaM rescue. Data are shown as fold of change (normalized to the control). Downloaded from www.jbc.org at UMDNJ RW JOHNSON, on June 4, 2012

Supplementary Table 2 Numeric data presented in the paper . Figure number Descriptions Mean ± SEM n p Value*

Figure 1 1E Syt1 KO mIPSC Frequency (Hz)

Control 7.46 ± 0.42 18

CaM KD 3.55 ± 0.45 21 P<0.001

CaM KD+WT CaM 6.59 ± 0.65 17 n.s.

1F Syt1 KO mIPSC Amplitude (pA) Downloaded from Control 26.5 ± 1.6 18

CaM KD 30.5 ± 1.8 21 n.s.

CaM KD+WT CaM 28.4 ± 2.7 17 n.s.

www.jbc.org 1G Syt1 KO mIPSC Rise Time (ms)

Control 1.92 ± 0.11 18

CaM KD 1.83 ± 0.11 21 n.s. at UMDNJ RW JOHNSON, on June 4, 2012

CaM KD+WT CaM 1.79 ± 0.12 17 n.s. 21.0222 2.8229 23.7286 0.6222 22.5706 0.7204

1G Syt1 KO mIPSC Decay Time (ms)

Control 21.02 ± 2.82 18

CaM KD 23.73 ± 0.62 21 n.s.

CaM KD+WT CaM 22.57 ± 0.72 17 n.s.

Figure 2 2B Syt1 KO evoked IPSC Amplitude (nA)

Control 0.24 ± 0.02 44

CaM KD 1.32 ± 0.13 49 P<0.001

CaM KD+WT CaM 0.21 ± 0.03 15 n.s.

Figure 4 4A Relative mRNA level

Syt2 (Control) 1.00 ± 0.00 5

Syt2 (CaM KD) 10.42 ± 3.12 5 P<0.05

Syt2 (CaM KD+WT Res. 0.92 ± 0.15 4 n.s.

Lrrtm3 (Control) 1.00 ± 0.00 3

Lrrtm 3 (CaM KD) 0.47 ± 0.02 3 P<0.001

S10

Figure number Descriptions Mean ± SEM n p Value*

CaM 3 (Control) 1.00 ± 0.00 4

CaM 3 (CaM KD) 0.26 ± 0.08 4 P<0.01

CaM 3 ((CaM KD+WT Res.) 0.10 ± 0.03 4 P<0.001

Figure 5 5B Protein expression level (%)

CaM (Control) 100 ± 13 3

CaM (CaM KD) 23 ± 3 3 P<0.01

CaM (CaM KD+WT Res) 200 ± 19 3 P<0.05

CaM (CaM KD+CaM1,2,3,4) 467 ± 54 3 P<0.01 Downloaded from

Syt2 (Control) 100 ± 39 3

Syt2 (CaM KD) 1045 ± 37 3 P<0.001 www.jbc.org Syt2 (CaM KD+WT Res) 107 ± 41 3 n.s.

Syt2 (CaM KD+CaM1,2,3,4) 401 ± 59 3 P<0.05

at UMDNJ RW JOHNSON, on June 4, 2012

Rab3A (Control) 100 ± 29 3 n.s.

Rab3A (CaM KD) 59 ± 21 3 n.s.

Rab3A (CaM KD+WT Res) 92 ± 27 3 n.s.

Rab3A (CaM KD+CaM1,2,3,4) 58 ± 9 3 n.s.

Syt1 (Control) 100 ± 5 3

Syt1 (CaM KD) 55 ± 15 3 P<0.05

Syt1 (CaM KD+WT Res) 102 ± 6 3 n.s.

Syt1 (CaM KD+CaM1,2,3,4) 44 ± 15 3 P<0.05

Synt1 (Control) 100 ± 16 3

Synt1 (CaM KD) 97 ± 28 3 n.s.

Synt1(CaM KD+WT Res) 85 ± 10 3 n.s.

Synt1 (CaM KD+CaM1,2,3,4) 75 ± 9 3 n.s.

SNAP25 (Control) 100 ± 19 3

SNAP25 (CaM KD) 87 ± 35 3 n.s.

SNAP25 (CaM KD+WT Res) 101 ± 14 3 n.s.

SNAP25 (CaM KD+CaM1,2,3,4) 64 ± 22 3 n.s.

Rabphilin (Control) 100 ± 16 3

S11

Figure number Descriptions Mean ± SEM n p Value*

Rabphilin (CaM KD) 36 ± 5 3 P<0.05

Rabphilin (CaM KD+WT Res) 126 ± 14 3 n.s.

Rabphilin (CaM KD+CaM1,2,3,4) 31 ± 2 3 P<0.05

CSP (Control) 100 ± 2 3

CSP (CaM KD) 75 ± 11 3 n.s.

CSP (CaM KD+WT Res) 90 ± 17 3 n.s.

CSP (CaM KD+CaM1,2,3,4) 56 ± 13 3 P<0.05

Munc 18 (Control) 100 ± 29 3 Downloaded from

Munc 18 (CaM KD) 69 ± 21 3 n.s.

Munc 18 (CaM KD+WT Res) 93 ± 22 3 n.s.

Munc 18 (CaM KD+CaM1,2,3,4) 92 ± 32 3 n.s. www.jbc.org

SCAMP (Control) 100 ± 32 3

SCAMP (CaM KD) 60 ± 16 3 n.s. at UMDNJ RW JOHNSON, on June 4, 2012

SCAMP (CaM KD+WT Res) 113 ± 29 3 n.s.

SCAMP (CaM KD+CaM1,2,3,4) 73 ± 23 3 n.s.

PSD95 (Control) 100 ± 6 3

PSD95 (CaM KD) 81 ± 1 3 P<0.05

PSD95 (CaM KD+WT Res) 90 ± 7 3 n.s.

PSD95 (CaM KD+CaM1,2,3,4) 76 ± 7 3 P<0.05

NSF (Control) 100 ± 5 3

NSF (CaM KD) 69 ± 1 3 P<0.01

NSF (CaM KD+WT Res) 95 ± 8 3 n.s.

NSF (CaM KD+CaM1,2,3,4) 78 ± 6 3 n.s.

Neuroligin 1 (Control) 100 ± 25 3

Neuroligin 1 (CaM KD) 70 ± 15 3 n.s.

Neuroligin 1 (CaM KD+WT Res) 85 ± 14 3 n.s.

Neuroligin 1 (CaM KD+CaM1,2,3,4) 74 ± 19 3 n.s.

Neuroligin 2 (Control) 100 ± 20 3

Neuroligin 2 (CaM KD) 88 ± 6 3 n.s.

Neuroligin 2 (CaM KD+WT Res) 95 ± 7 3 n.s.

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Figure number Descriptions Mean ± SEM n p Value*

Neuroligin 2 (CaM KD+CaM1,2,3,4) 84 ± 5 3 n.s.

Figure 6 6B CaM/VCP

Cortex 1.66 ± 0.04 4

Cerebellum 1.16 ± 0.06 4 P<0.001

Spinal cord 0.85 ± 0.06 4 P<0.001

Syt1/VCP

Cortex 6.97 ± 0.12 4

Cerebellum 2.55 ± 0.26 4 P<0.001 Downloaded from

Spinal cord 1.05 ± 0.15 4 P<0.001

Syt2/VCP www.jbc.org Cortex 0.46 ± 0.02 4

Cerebellum 1.46 ± 0.03 4 P<0.001

Spinal cord 1.04 ± 0.04 4 P<0.001 at UMDNJ RW JOHNSON, on June 4, 2012

Syb1/VCP

Cortex 0.73 ± 0.04 4

Cerebellum 2.77 ± 0.09 4 P<0.001

Spinal cord 2.33 ± 0.20 4 P<0.001

Figure 7 7D Rescue evoked IPSCs Amplitudein Syt1 KO

Control (same data set as in 1b) 0.24 ± 0.02 44

CaM KD+CaM1,2 0.22 ± 0.04 17 n.s.

CaM KD+CAM3,4 0.82 ± 0.12 13 P<0.001

CaM KD+CaM1,2,3,4 0.95 ± 0.18 16 P<0.001 * P value obtained by Student t-test, compared to control group.

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A 48 hour 72 hour 96 hour B

T286D

Control TTX APV NifedipineControl TTX APV NifedipineControl TTX APV CaM KDControl Nifedipine

Control CaM KD α VCP KD+CaMKIIKD+CaMKIIα Syt2 Syt2

Syt1 Synt. Synt. Downloaded from

Suppl. Figure 1 Pang et al. www.jbc.org at UMDNJ RW JOHNSON, on June 4, 2012