Calmodulin Suppresses Synaptotagmin-2 Transcription inCortical 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. Sudhof‡§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 thatplays a pivotal role in regulating innumerable neuronal func-tions, including synaptic transmission. In cortical neurons,most neurotransmitter release is triggered by Ca2� binding tosynaptotagmin-1; however, a second delayed phase of release,referred to as asynchronous release, is triggeredbyCa2�bindingto an unidentified secondary Ca2� sensor. To test whether CaMcould be the enigmatic Ca2� sensor for asynchronous release,we now use in cultured neurons short hairpin RNAs that sup-press expression of �70% of all neuronal CaM isoforms. Sur-prisingly, we found that in synaptotagmin-1 knock-out neurons,the CaM knockdown caused a paradoxical rescue of synchro-nous release, instead of a block of asynchronous release. Geneand protein expression studies revealed that both in wild-typeand in synaptotagmin-1 knock-out neurons, the CaM knock-downaltered expression of>200 genes, including that encodingsynaptotagmin-2. Synaptotagmin-2 expression was increasedseveral-fold by the CaM knockdown, which accounted for theparadoxical rescue of synchronous release in synaptotagmin-1knock-out neurons by the CaM knockdown. Interestingly, theCaM knockdown primarily activated genes that are preferen-tially expressed in caudal brain regions, whereas it repressedgenes in rostral brain regions. Consistent with this correlation,quantifications of protein levels in adult mice uncovered aninverse relationship of CaM and synaptotagmin-2 levels inmouse forebrain, brain stem, and spinal cord. Finally, weemployed molecular replacement experiments using a knock-down rescue approach to show that Ca2� binding to the C-lobebut not the N-lobe of CaM is required for suppression of synap-totagmin-2 expression in cortical neurons. Our data describe apreviously unknown, Ca2�/CaM-dependent regulatory path-way that controls the expression of synaptic proteins in the ros-tral-caudal neuraxis.

Neurotransmitter release is mediated by two separate, com-peting pathways: synchronous and asynchronous releases. Both

modes of synaptic vesicle exocytosis are triggered by Ca2�. Thesynchronous release mode exhibits an apparent Ca2� cooper-ativity of �5 (1–3), and the asynchronous release shows anapparent Ca2� cooperativity of �2 (3). The role of synaptotag-mins as primary Ca2� sensors for synchronous neurotransmit-ter release is well established (4–11). However, the molecularidentity of the Ca2� sensor that mediates asynchronous releaseremains unknown.Calmodulin (CaM)4 is a ubiquitous and essential Ca2�-bind-

ing protein that regulates a plethora of cellular processes, fromgene transcription to signal transduction to ion channels tomembrane traffic (12–14). CaM is highly conserved in verte-brates and is ubiquitously expressed. All CaM proteins arecomposed of two lobes (i.e. the N- and C-lobes) that each con-tain two E-F hand Ca2�-bindingmotifs and are connected via aflexible �-helix (15). Each E-F hand motif binds to one Ca2�

ion. Ca2� binds to the N- and C-lobes in a cooperative manner,with the N-lobe binding Ca2� with a lower affinity but fasterassociation and dissociation rates than the C-lobe (16, 17). Thedifferent Ca2� binding characteristics probably confer ontoCaM lobes specific target protein binding properties and func-tions (18, 19). Apart from numerous cytoplasmic regulatoryfunctions, Ca2� binding toCaM serves to activate transcriptionby a number of distinct signaling pathways (14, 20).CaM regulates neurotransmitter release by multiple mecha-

nisms, including binding toMunc13, regulatingCa2� channels,and activating Ca2�/CaM-dependent kinase II (CaMKII) (12–14, 20–24). In addition, CaMwas proposed to directly functionas a Ca2� sensor for Ca2�-triggered exocytosis (25, 26),prompting us to test here whether CaM may act as the Ca2�

sensor for asynchronous release. For this purpose, we culturedcortical neurons from synaptotagmin-1 (Syt1) knock-out (KO)mice in which synchronous release is abolished and only asyn-chronous release remains (5, 6).We then analyzed the effects ofshRNA-mediated knockdown (KD) of all CaM isoforms onneurotransmitter release, using a previously established lenti-viral system that suppresses�70% of neuronal CaM expression(24). We found that although KD of CaM had no significanteffect on asynchronous release, it surprisingly rescued the lossof synchronous release in Syt1 KO neurons. An unbiasedgenome-wide gene expression profiling experiment revealedthat the CaM KD induced a dramatic up-regulation of expres-

* This work was supported, in whole or in part, by a National Institutes of MentalHealth Grant (to T. C. S.). This work was also supported by awards from NationalAlliance for Research on Schizophrenia and Depression (to Z. P. P.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables S1 and S2 and Fig. S1.

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, CA94304-5543. Tel.: 650-721-1421; E-mail: zpang@stanford.edu.

3 To whom correspondence may be addressed: Dept. of Molecular andCellular Physiology, Stanford University, 1050 Arastradero Rd., PaloAlto, CA 94304-5543. Tel.: 650-721-1421; E-mail: tcs1@stanford.edu.

4 The abbreviations used are: CaM, calmodulin; CaMKII, CaM-dependentkinase II; Syt, synaptotagmin; KO, knock-out; Syb, synaptobrevin; DIV,day(s) in vitro; mIPSC, miniature inhibitory postsynaptic currents; VCP,vasolin-containing protein.

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.

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sion of Syt2 and synaptobrevin-1 (Syb1), which are normallyexpressed in the forebrain at low levels but are abundant incaudal brain regions (27–29). In addition, the expression ofother caudal synaptic genes was increased, whereas expressionof rostral synaptic genes was decreased. Moreover, usingmolecular replacement experiments, we show that the regula-tion of Syt2 expression by CaM requires Ca2� binding to onlytheC-lobe but not theN-lobe of CaM. Thus, our data show thatCa2� binding to CaM regulates neurotransmitter release notonly in the short term by binding to target proteins (12–14,20–24) but also on a longer time frame by modulating theexpression of presynaptic proteins such as Syt2, thereby influ-encing the properties of neurotransmitter release at a synapse.

EXPERIMENTAL PROCEDURES

Neuronal Culture—Mouse cortical culture was made asdescribed elsewhere (6, 24). Briefly, the primary cortical neu-ronswere isolated frompostnatal day 0 pups of Syt1 deficient orwild-type mice, dissociated by papain digestion, and plated onMatrigel-coated circle glass coverslips. The neurons were cul-tured in vitro for 13–16 days in minimal essential medium(Invitrogen) supplemented with B27 (Invitrogen), glucose,transferrin, fetal bovine serum, and Ara-C (Sigma).Lentivirus Packaging and Infection of Neuronal Culture—The

packaging of lentiviruses and the infection of neurons with len-tiviruses were described previously (24). Briefly, the lentiviralexpression vector (control vector L309 or the shRNAs carryingvectors) and three helper plasmids, the pRSV-REV, pMDLg/pRRE, and vesicular stomatitis virus G protein were co-trans-fected into HEK 293T cells (ATCC, Manassas, VA) at 6, 2, 2,and 2 �g of DNA/25-cm2 culture area, respectively. The tran-sient transfections were performed with FuGENE 6 transfec-tion reagent (Roche Applied Science) following the manufac-turer’s instructions. Supernatants with viruses were collected48 h after transfection. Cortical neuronal culture was infectedat 5 days in vitro (DIV) and used for biochemical or physiolog-ical analysis on 14–16 DIV. All of the steps were performedunder level II biosafety conditions.Lentiviral Vector Construction—Lentiviral vectors construc-

tions were described previously (24). Human H1 promoter andhuman U6 promoter were cloned into lentiviral backbone vec-tor FG-12 vector. Cloning sites after H1 promoter are XhoI-XbaI-HpaI; cloning sites after U6 promoter are AscI-ClaI-Rs-rII-PacI. Internal ribosome entry site-enhanced GFP wascloned in after ubiquitin C promoter, leaving BamHI-EcoRIsites for inserting rescue cDNAs. Short hairpin sequences forCaMs were the same as described previously (24).Microarray Expression Assays—The cultured cortical neu-

rons were lysed, and total RNAwas extracted and purified witha RNAqueousmicro kit (Ambion) following themanufacturer’sinstructions. Standard gene expression analyses were per-formed using the Affymetrix mouse gene ST_1.0 chip by theProtein and Nucleic Acid Facility at Stanford University. Arraydata were analyzed using the Partek genomics suite. Geneexpression levels were compared with their control groupsindividually. Two data sets of CaM KD and CaM KD � WTCaM rescue were obtained with two batches of cultured neu-rons infected with control viruses; viruses with shRNAs target-

ingCaM1–3; or viruses with shRNAs and expression of shRNAsilent WT CaM cDNA (supplemental Table S1). Only the geneexpression levels changed after CaM KD and rescued withWTCaM in both experiments are included in the list. Full geneexpression array data are deposited to the NCBI Gene Expres-sion Omnibus.Quantification of mRNA Level by Quantitative Real Time

PCR—The cultured cortical neurons were lysed, and total RNAwas extracted and purified with a RNAqueous micro kit(Ambion) following the manufacturer’s instructions. ThemRNA level of individual genes was then analyzed by one-stepquantitative real time PCR system with pre-made TaqMan�gene expression assays (Applied Biosystems). Briefly, 30 ng ofRNA sample in 1�l of volumewasmixedwith 10�l of TaqManfast universal PCR master mix (twice), 0.1 �l of reverse tran-scriptase (50 units/�l), 0.4 �l of RNase inhibitor (20 units/�l),7.5 �l of H2O, and 7 �l of TaqMan� gene expression assay forthe target gene (including the forward and reverse primers andthe TaqMan FAM-MGB probe). The reaction mixture wasloaded onto ABI7900 fast real time PCR machine for 30 min ofreverse transcription at 48 °C followed by 40 PCR amplificationcycles consisting of denaturation at 95 °C for 1 s and annealingand extension at 60 °C for 20 s. The amplification curve wascollected and analyzed with ��Ct methods for relative quanti-fication of mRNAs. The amount of mRNA of target genes, nor-malized to that of an endogenous control and relative to thecalibrator sample, is calculated by 2���Ct. In the current study,GAPDH was used as the endogenous control, and the RNAsamples derived from neurons infected with control vector(L309) were used as calibrators. The TaqMan� gene expres-sion assays (Applied Biosystems) used in the current studyincluded: Mm00486655_m1 (CaM 1), Mm00849529_g1 (CaM2), Mm00482929_m1 (CaM 3), Mm00618457_m1 (Lrrtm3),Mm00436864_m1 (Syt2), and mouse GAPD (GAPDH) endog-enous control. Syt9 quantitative real time PCR PrimeTimeassay was designed and custom-made through Integrated DNATechnologies.Electrophysiology—Electrophysiology was performed as

described previously (6, 24, 30). Briefly, the evoked synapticresponses were triggered by a bipolar electrode (FHC,CBAEC75 Concentric Bipolar Electrode OP: 125 �m SS; IP: 25�m Pt/lr) placed at a position 100–150 �m from the soma ofneurons recorded. The patch pipettes were pulled from borsili-cate glass capillary tubes (Warner Instruments; catalog number64-0793) using PP830 or PC-10 pipette puller (Narishige). Theresistance of pipettes filled with intracellular solution variedbetween 4 and 5 MOhm. After formation of whole cell config-uration and equilibration of intracellular pipette solution, theseries resistance was adjusted to 8–10 MOhm. Synaptic cur-rents were monitored withMulticlamp 700B amplifier (Molec-ularDevices). The frequency, duration, andmagnitude of extra-cellular stimulus were controlled with a model 2100 isolatedpulse stimulator (A-M Systems, Inc.) synchronized withClampex 9 data acquisition software (Molecular Devices). Thewhole cell pipette solution contained 135 mM CsCl, 10 mM

HEPES, 1mMEGTA, 1mMNa-GTP, 4mMMg-ATP, and 10mM

QX-314 (pH 7.4, adjusted with CsOH). The bath solution con-tained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10

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mM HEPES, and 10 mM glucose (pH 7.4, adjusted with NaOH).Inhibitory and excitatory postsynaptic currents were pharma-cologically isolated by adding AMPA and NMDA receptorblockers 6-cyano-7-nitroquinoxaline-2,3-dione (20 �M) andAP-5 (50 �M) or GABAA receptor blockers bicuculine (20 �M)or picrotoxin (50 �M) to the extracellular bath solution. Spon-taneous miniature inhibitory postsynaptic currents (mIPSCs)weremonitored in the presence of tetrodotoxin (1 �M) to blockthe action potentials.Miscellaneous Procedures—For immunoblotting analyses

from cultured neurons, at 14–15 DIV, the cultures were

washed twice using PBS. The mate-rials were directly collected by SDSprotein sample buffers (50 �l ofsample buffer/well for 24-wellplates). Equal amounts of samples(25�l) were analyzed by SDS-PAGEand immunoblotting using anti-bodies as follows: CaM monoclo-nal 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 carriermembrane proteins (R806, 1:1,000);PSD95 (L667, 1:1,000); Munc18(J371, 1:1,000); cysteine string pro-tein (R807, 1:1,000); N-ethyl-maleimide-sensitive factor (P944,1:1,000); NL1 (4C12, 1:1,000); NL2(169C, 1:200); GDP dissociationinhibitor (81.2, 1:2,000); synaptot-brevin-1 (P938, 1:500); �-actin(mouse monoclonal antibody clone14; BD Transduction Labs; 1:2,000);and vasolin-containing protein(VCP; K330, 1:1,000). For proteinquantitations, 125I-labeled second-ary antibodies and PhosphoImagerdetection (Molecular Dynamics)were used. GDP dissociation inhib-itor and VCP were employed asinternal standards.Data Analysis—The electro-

physiological currents were sam-pled at 10 kHz and analyzed off-line using Clampfit 9 (MolecularDevices) software. For graphic rep-resentation of the current tracesshown for evoked synaptic trans-mission, stimulus artifacts wereremoved. For measurements of fre-quency of spontaneous release andamplitudes of synchronous IPSCsduring stimulus trains, individualmIPSCs or IPSCs were collectedusing pClamp template search func-

tion. Cumulative distributions of inter-event interval andamplitude of mIPSCs were compared using a Kolmogorov-Smirnov test. All of the statistical comparisonsweremadeusingStudent’s t test except where otherwise stated.

RESULTS

CaM KD Rescues Synchronous Release in Syt1 KO Synapses—We used shRNAs targeting all CaM isoforms (24) to suppressCaM expression in cultured cortical neurons from Syt1 KOmice. Immunoblotting analysis with enhanced chemilumines-cence detection suggested that the CaM KD strongly sup-

FIGURE 1. CaM knockdown reduces mini release in Syt1 KO neurons. Cultured cortical neurons from Syt1KO mice were infected with control lentivirus or with CaM KD lentivirus expressing CaM shRNAs without or withwild-type CaM rescue mRNA (�WT CaM) (24). Neurons were cultured from newborn mice, infected at DIV 5,and analyzed at DIV 14 –15. A, representative immunoblots of neurons probed with antibodies to CaM, Syt1,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 fromfive neurons. p � 0.001 for control versus CaM KD; the values for control versus CaM KD with rescue were notsignificant. A Kolmogorov-Smirnov test was used. D, cumulative distributions of the mIPSC amplitudes. Theplot shows averages of minis from five neurons. The values for control versus CaM and control versus CaM KDwith rescue were not significant. A Kolmogorov-Smirnov test was used. E, summary graphs of the mIPSCfrequency. F, summary graphs of the mIPSC amplitude. G, summary graphs of the 10 –90% rise time of mIPSCs.H, summary graphs of the 90% to 10% decay time of mIPSCs. The data shown are the means � S.E.; n � numberof cells indicated in the bars from three independent cultures. ***, p � 0.001 as assessed with Student’s t test.

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pressesCaMexpression (Fig. 1A), and quantitations of theCaMmRNA and protein levels confirmed an actual suppression ofCaM expression by �70% (24).Syt1 functions not only as a Ca2� sensor for synchronous

release but also for spontaneous miniature synaptic release(“minis”); it additionally acts as a clamp formini release (31). Asa result, the Syt1 KO causes a large increase in the frequency ofminis; however, the increased minis in Syt1-deficient synapsesremain Ca2�-sensitive and are likely mediated by a secondary,as yet unidentified Ca2� sensor that exhibits the same proper-ties as the Ca2� sensor for asynchronous release. Thus, we firsttestedwhether theCaMKDalters the increasedminis observedin the absence of Syt1. Indeed, we found that the CaM KDreduced the frequency ofmini IPSCs in Syt1 KOneurons�40%(Fig. 1, B, C, and E). This reduction in mini release was fullyrescued by expression of shRNA-resistant wild-typeCaMusingthe same lentivirus (24), demonstrating that themini reductionis not a result of an off-target effect of the shRNAs used for theexperiment. No obvious changes have been observed in theamplitudes ofmIPSCs afterCaMKD (Fig. 1,D and F) and kinet-ics of mIPSCs (Fig. 1, G and H).We next examined evoked asynchronous release in Syt1 KO

cultured neurons with or without rescue. To our surprise, we

found that instead of impairingasynchronous release, the CaM KDreversed the loss of synchronousrelease in Syt1 KO neurons (Fig. 2,A–C). Analysis of the kinetics ofevoked responses revealed thatupon CaM KD, the massivelydelayed release reaction in Syt1 KOsynapses is accelerated to wild-typelevels (Fig. 2C). Again, this CaMKDphenotype could be fully rescuedby expression of wild-type CaM.Moreover, the restoration of syn-chronous release is also evident dur-ing trains of stimulation (Fig. 2D).However, the presence of synapticfacilitation instead of depressionduring the stimulus trains indicatedthat the release probability of CaMKD synapses was lower than that ofWT synapses (Fig. 2E), consistentwith our previous observation thatthe CaMKD decreases the presynap-tic release probability by a CaMKII-dependent mechanism (24).Gene Expression Profiling Identifies

Multiple Synaptic CaM Targets—To search for a potential mecha-nism that accounts for the rescue ofsynchronous release by theCaMKDin Syt1 KO neurons, we performedan unbiased gene expression analy-sis in cortical neurons. To avoidartifacts induced by the Syt1 KO orby off-target effects, we used wild-

type neurons and directly compared neurons that had beeninfected with lentiviruses expressing the CaM shRNAs eitherwithout or with a wild-type CaM rescue protein. We then ana-lyzed the gene expression patterns in these neurons with theAffymetrixmouse gene ST_1.0 chip.We identified in two inde-pendent array studies �250 genes whose expression was con-sistently up- or down-regulated by the CaM KD, as comparedwith the CaM KD/rescue control (see Fig. 4; Table 1 and sup-plemental Table S1; deposited to the NCBI Gene ExpressionOmnibus.As expected, multiple classes of genes were regulated by

CaM. Consistent with previous studies (32, 33), we found thatactivity-dependent genes, such as Homers, Npas2, Arc, andEgr3 (supplemental Table S1), were down-regulated by theCaM KD. Interestingly, we observed that several synaptic traf-ficking proteins were either up- or down-regulated by the CaMKD (Fig. 3 and supplemental Table S1). Among these was alarge increase in the expression of Syt2, which can serve as aCa2� sensor for synaptic exocytosis (3, 7–9); thus, this up-reg-ulation of Syt2 by the CaM KD likely accounts for the rescue ofthe Syt1 KO phenotype. In addition, expression of Syb1 wasmassively increased, whereas expression of Syt4, Syt9, and syn-taxin-1A was decreased. Another intriguing class of proteins

FIGURE 2. CaM KD restores synchronous release in Syt1 KO neurons. Cultured cortical neurons from Syt1 KOand littermate wild-type mice were infected with control lentivirus or with CaM KD lentivirus expressing CaMshRNAs without or with wild-type CaM rescue mRNA (24). Neurons were cultured from newborn mice, infectedat DIV 5, and analyzed at DIV 14 –15. A, representative traces of evoked IPSCs in Syt1 KO neurons. B, summarygraphs of the peak amplitudes of evoked IPSCs (means � S.E.; numbers of neurons analyzed are shown in bars;n � 3 independent cultures; ***, p � 0.001 per Student’s t test). C, time course of the synaptic IPSC chargetransfer induced by isolated action potentials. The curves are the averages of n � 11 neurons in each group.D, representative traces of IPSCs evoked by 10 stimuli at 10 Hz in control or CaM KD Syt1 KO neurons and inwild-type neurons. E, normalized synchronous IPSC amplitudes during the 10 Hz stimulus train in CaM KD Syt1KO (n � 22) and wild-type (n � 10) neurons. The data are the means � S.E.; the numbers in the bars indicate thenumber of cells analyzed in at least three independent experiments; statistical significance was calculated byStudent’s t test (p � 0.01). Numerical data are listed in supplemental Table S2.

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whose expression was strongly regulated by CaM were celladhesion molecules, such as the synaptic cell adhesion mole-cules Lrrtm1, Lrrtm3, and contactin-2 (Fig. 3). Moreover, weobserved up-regulation of sodium channels, and a down-regu-lation of potassium channels, suggesting that CaM might con-trol the activity-dependent regulation of neuronal excitability.Finally, we detected changes in multiple genes encoding tran-scription factors, intracellular signal transduction proteins, ele-ments of the cytoskeleton, ormetabolic enzymes (supplementalTable S1). It should be noted, however, that despite these mul-tifarious changes, more than 95% of genes showed no CaMKD-induced change, suggesting that the observedCaMKD-de-pendent expression changes are specific.Validation of Microarray Results by Quantitative Real Time

PCR and Immunoblotting—We validated the microarrayresults by quantification of themRNAs for three representativegenes. Quantitative real time PCR measurements confirmedthat Syt2 expression, tested because of its Ca2� sensor function,was up-regulated �10-fold by the CaM KD, whereas Lrrtm3and Syt9 expression were down-regulated �2-fold (Fig. 4A). Inaddition, becausewe are employing a rat CaM2 cDNA to rescuethe mouse CaM KD phenotype and because the TaqMan�assays for mouse CaM3 that we employed to measure themRNA levels do not detect the rat CaMmRNA, wewere able touse quantitative real time PCR to confirm the CaM KD effi-

ciency even under rescue conditions. CaM mRNA levels werelow in CaM KD samples and remained low even under rescueconditions (Fig. 4A). Thus, our results indicate that the lentivi-rally mediated KD of CaM is very effective in cultured neurons.Next, we analyzed the expression of selected proteins

encoded by the mRNAs that were altered by the CaM KD.Immunoblotting confirmed that the CaM KD produced astrong induction of Syt2 and Syb1 protein, consistent with themicroarray data (Fig. 4B). Syt2 and Syb1 were expressed at verylow levels in control cortical cultures that only expressenhanced GFP; however, in CaMKD condition we found obvi-ous expression of both Syt2 and Syb1 (Fig. 4B). Again, the up-regulation of Syt2 and Syb1 can be reduced (rescued) by over-expression of wild-type CaM in CaM KD neurons.To achieve amore quantitative understanding of the changes

in protein expression upon CaMKD, wemeasured the levels of14 synaptic proteins in the CaM knockdown neurons usingquantitative immunoblotting with 125I-labeled secondary anti-bodies and PhosphoImager detection. In this analysis, we notonly analyzed neurons infected with control and CaM KD len-tiviruses but also neurons in which the CaM KD viruses addi-tionally produced shRNA-resistant mRNAs encoding eitherwild-type rat CaM or mutant rat CaM that contains mutationsin all four Ca2�-binding sites (called CaM1,2,3,4). These mea-surements further confirmed the array data, demonstrating a

TABLE 1Correlation of gene expression changes induced by the CaM KD in cortical neurons with the rostral-caudal expression patterns of these genesas deduced from the Allen Brain AtlasArray 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 uparrow 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 arrowindicates 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 asfollows: the average values of medulla and pons: the average values of cortex and hippocampus.

Gene name Description Expression levelin CaM KD (microarray)

-Fold change(CaM KD/control)

Rostral to caudalexpression level

(Allen Brain Atlas)Ratio

(caudal/rostral)

Syt2 synaptotagmin 2 1 5.64 3 3.04Cntn2 contactin 2 1 2.22 3 2.02Lgi3 leucine-rich repeat LGI family, member 3 1 2.12 3 1.12Coro6 coronin, actin binding protein 6 1 3.96 3 2.06Flt3 FMS-like tyrosine kinase 3 1 2.56 3 2.79Phospho1 phosphatase, orphan 1 1 2.15 3 4.11Aldh1a7 aldehyde dehydrogenase family 1, subfamily A7 1 1.79 3 2.35Tspan17 tetraspanin 17 1 1.71 3 1.44Tspan2 tetraspanin 2 1 1.75 3 1.26F3 coagulation factor III 1 1.46 3 2.90Stx1a syntaxin 1A 2 0.61 4 0.30Icam5 intercellular adhesion molecule 5 2 0.54 4 0.02Lrrtm1 leucine-rich repeat transmembrane neuronal 1 2 0.69 4 0.05Opcml opioid-binding protein 2 0.6 4 0.37Lrrtm3 leucine-rich repeat transmembrane neuronal 3 2 0.62 4 0.47Npas2 Neuronal PAS domain protein 2 2 0.65 4 0.14NueroD6 neurogenic differentiation 6 2 0.38 4 0.46Arpc5 actin-related protein 2 2 0.63 4 0.11Mtap9 microtubule-associated protein 9 2 0.67 4 0.54Dlg3 discs, large homolog 3 2 0.64 4 0.25Necab1 N-terminal EF-hand calcium binding protein 1 2 0.61 4 0.27Doc2B double C2 domain protein B 2 0.58 4 0.18Cpne5 copine 5 2 0.56 4 0.23Sez6 Seizure related gene 6 2 0.63 4 0.34Ypel2 Yipppee-like 2 2 0.6 4 0.57Galnt9 UDP-N-acetyl-a-D-galactosamine 2 0.58 4 0.52Tiam2 T-cell lymphoma invasion and metastasis 2 2 0.62 4 0.14Ctxn1 cortexin 1 2 0.6 4 0.20Pak7 p21 (CDKN1A)-activated kinase 7 2 0.58 4 0.07Rab40b Rab40b, member RAS oncogene family 2 0.62 4 0.30Rimbp2 RIMS binding protein 2 2 0.52 4 0.72Tmem74 transmembrane protein 74 2 0.55 4 0.25Lingo1 leucine-rich repeat and Ig domain containing 1 2 0.56 4 0.22Epha6 Eph receptor A6 2 0.56 4 0.62Ephb6 Eph receptor B6 2 0.69 4 0.45

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large increase in Syt2 levels induced by the CaM knockdown(�10-fold), and additionally revealed changes inmultiple otherproteins, especially in rabphilin, whose levels decreased �60%(Fig. 5). Note that in this analysis the lack of rescue by mutantCaM1,2,3,4 that is unable to bindCa2� provides a further controlthat ensures the specificity of the results.CaM-regulated Gene Expression Correlates with a Rostral-

Caudal Gradient—Inspection of the gene expression changesinduced by CaM suggests that the genes that are up-regulatedby the CaM KD, such as Syt2 and Syb1, are preferentiallyexpressed in caudal brain regions, whereas genes that are

down-regulated, such as Syt9 and Lrrtms, are primarilyexpressed in rostral brain regions. Indeed, quantitation of theratio of expression of selected genes in rostral versus caudalbrain regions using the Allen Brain Atlas reveals a tight corre-lation of the direction of the gene expression change induced bythe CaM KD and the rostral-caudal expression gradient of agene (Table 1). This suggests that although not all genesexpressed in a rostral-caudal gradient are subject to CaM reg-ulation in cultured cortical neurons, those whose expression isregulated by CaM in this system are expressed in a predictablegradient in the brain.These observations led us to hypothesize that possibly CaM

itself is expressed in a rostral-caudal gradient in brain and maycontribute to the specification of rostral-caudal gene expres-sion.We thusmeasured the levels of CaM, Syt1, Syt2, Syb1, andVCP (as a load control) in the cortex, cerebellum, and spinalcord of four adult mice by quantitative immunoblotting. Strik-ingly, we found that CaM was expressed in a rostral-caudalgradient, 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 andSyb1 displayed an inverse rostral-caudal gradient (Fig. 6).CaM Control of Syt2 Expression Is Independent of CaMKII�—

The Ca2� dependence of the CaM-mediated suppression ofSyt2 expression prompted us to ask whether Syt2 expression incultured neurons is activity-dependent. However, inhibition ofneuronal activity in cultured cortical neurons using tetrodo-toxin (a Na� channel inhibitor), AP-5 (an NMDA receptorinhibitor), or nifedipine (an L-type Ca2� channel blocker) hadno significant effect on Syt2 expression (supplemental Fig. S1).This result is consistent with the notion that the Ca2�/CaM-dependent control of Syt2 expression does not operate on ashort term basis in neurons but is a developmental process.We previously found that in presynaptic terminals, one path-

way by which CaM controls synaptic strength is through theactivation of CaMK II (24). Because in T lymphocytes, CaMactivates CaMKII� to inhibit IL2 gene expression (34, 35), wetested whether overexpression of a constitutively activemutantof CaMKII� (CaMKII�T286D) reverses the activation of Syt2gene expression upon CaM KD. The rationale for this experi-ment is that the same CaMKII� mutant rescues the decrease insynaptic strength induced by CaM KD (24), suggesting thatit may represent a general pathway of CaM action. How-ever, immunoblotting demonstrated that neither wild-typeCaMKII� nor constitutively active mutant CaMKII�T286D

reversed the increase in Syt2 expression induced by the CaMKD (supplemental Fig. S1B), indicating that the suppression ofSyt2 gene expression by CaM is independent of CaMKII�.Ca2� Binding to the C-lobe of CaM Sufficed to Suppress Syt2

Expression—CaM suppression of Syt2 expression in culturedcortical neurons requires Ca2� binding, because the CaMmutant in which all four Ca2�-binding sites were abolished(CaM1,2,3,4) was unable to rescue the CaM KD phenotype (Fig.5). The two lobes of CaM, the C- andN-lobes, each contain twoE-F hand Ca2�-binding sites. Because studies on CaM-regu-lated ion channels uncovered a differential requirement forCa2� binding to the N- or C-terminal lobes of CaM (36, 37), we

FIGURE 3. Profiling of gene expression in control, CaM KD, or CaM KD withWT CaM rescue. Cultured cortical neurons from newborn wild-type micewere infected at DIV 5 with control lentivirus or with CaM KD lentivirusexpressing CaM shRNAs without or with wild-type CaM rescue mRNA andanalyzed using Affimetrix arrays at DIV 14 –15. A, heat map plot of geneexpression changes in CaM KD neurons without or with wild-type CaM res-cue, as compared with control neurons. Only genes with CaM KD-inducedchanges that were rescued by wild-type CaM are shown (n � 2 independentexperiments). B, functional classification of genes changed by the CaM KDand rescued by wild-type CaM.

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askedwhether all CaMCa2�-binding sites are required for sup-pression of Syt2 expression or whether Ca2� binding to one ofthe two lobes is sufficient. Strikingly, mutant CaM in whichCa2� binding to the N-terminal lobe of CaM was blocked still

fully rescued the increased Syt2expression induced by the CaMKD,whereas mutant CaM lacking Ca2�

binding to the C-terminal lobe wasinactive (Fig. 7). Thus, CaM nor-mally suppresses Syt2 expressionvia Ca2� binding to only its C-ter-minal lobe.

DISCUSSION

In this study, we tested whetherCa2� binding to CaM triggers asyn-chronous neurotransmitter release,prompted by the similarities be-tween the deduced properties of theasynchronous release Ca2� sensor(3) and CaM (15–17) and by previ-ous suggestions thatCaMmay func-tion as a Ca2� sensor of vesicle exo-cytosis (25, 26). As an approach, weused cultured cortical neurons fromneonatal mice and lentivirally deliv-ered shRNAs that efficiently sup-press expression of all CaM iso-forms in neurons (Fig. 1A). Ourinitial experiments focused on neu-rons from Syt1 KO in which syn-chronous release is deleted; thus,asynchronous release can be studiedin isolation in these neurons, andchanges in asynchronous release areeasily detected. To our surprise,however, we found that the CaMKD did not decrease asynchronousrelease measurably but insteadpartly rescued the loss of asynchro-nous release in the Syt1 KOneurons(Figs. 1 and 2). Thus, CaM is notthe Ca2� sensor for asynchronousrelease but instead normally sup-presses a pathway of synchronousrelease in Syt1 KO neurons that isredundant with Syt1.In our experimental paradigm

investigating cultured neurons fromnewborn mice, we studied not onlythe workings of synapses but alsothe maturation of neurons. In theperiod between the culture and len-tiviral infection of the neurons andtheir analysis 2 weeks later, the neu-rons developed from immature cellsto morphologically and functionallyadvanced neurons; at the time of

plating, the neurons lacked dendrites, axons, spines, and syn-apses, whereas at the time of analysis, all of these haddeveloped.Thus, we hypothesized that the CaM KD may have activatedsynchronous release in Syt1 KO neurons by inducing changes

FIGURE 4. Validation of gene expression in CaM KD by quantitative real time PCR and immunoblotting.Cultured cortical neurons from newborn wild-type mice were infected at DIV 5 with control lentivirus or withCaM 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 theCaM3 mRNA levels are not rescued by expression of the rescue CaM2 cDNA. B, immunoblotting analysis of Syt1,Syt2, CaM, and VCP (used as load control).

FIGURE 5. Quantitation of CaM KD-induced changes in protein levels. Cultured cortical neurons from new-born wild-type mice were infected at DIV 5 with control lentivirus or with CaM KD lentivirus expressing CaMshRNAs without a CaM mRNA or with either wild-type CaM rescue mRNA (� WT CaM) or a mutant CaM rescuemRNA (� CaM1,2,3,4) and analyzed at DIV 14. A, representative immunoblots of neuronal proteins using primaryantibody against the different proteins as indicated and 125I-conjugated secondary antibodies followed byPhosphoImager detection. B, summary graphs of protein levels measured by quantitative immunoblotting.The data shown are the means � S.E. *, p � 0.05; **, p � 0.01 as analyzed by Student’s t test (n � 3 independentcultures). 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.

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in gene expression. To explore this hypothesis, we performedwhole genome array studies that led to the identification of acohort of genes that were up- or down-regulated by the CaMKD (Fig. 3). Strikingly, Syt2 was among the up-regulated genes,and its activation by theCaMKDwas confirmedby quantitativemRNA and protein level measurements (Figs. 4 and 5). BecauseSyt2 normally acts as a fast Ca2� sensor for neurotransmitterrelease in hindbrain regions, such as the brain stem (3, 10), theincrease in Syt2 expression upon CaM KD provides a facileexplanation for the reversal of the Syt1 KO phenotype by theCaM KD. Note that in our experiments, all of the shRNA-de-pendent effects are controlled for by rescue experiments, anessential component given the many off-target effects ofshRNAs.The gene expression changes induced by the CaM KD were

relatively restricted, affecting only �250 genes (Fig. 3 and sup-plemental Table S1). Analysis of these gene expression changesyielded several observations. First, the expression of multiplegenes involved in neurotransmitter release was affected (e.g.Syb1, syntaxin-1A, and Syt9 in addition to Syt2); this suggeststhat themultifarious functions ofCaMat the synapse, functionsthat go beyond simply regulating ion channels, signal transduc-tion, and Munc13, include regulating synaptic gene expressionduring development. Second, apart from the expected changesin the expression of activity-dependent genes (e.g. Egr3, Npas2,Arc, and Homer) and of genes involved in intracellular signal-

ing and the cytoskeleton, the CaM KD also specifically alteredthe expression of cell adhesion molecules. We found that celladhesion molecules such as cntnap1 (contactin associate pro-tein 1, CASPR) were up-regulated, whereas other cell adhesionmolecules such as Lrrtms and latrophilins/CLs were down-reg-ulated by the CaM KD. Lrrtms have been shown to be the

FIGURE 6. Quantitative analysis of CaM, Syt1, Syt2, and Syb1 levels incortex, cerebellum, and spinal cord of mice. A, representative immuno-blots of mouse cortex, cerebellum/brain stem, and spinal cord homogenatesusing primary antibody against Syt1, Syt2, Syb1, and CaM and 125I-conju-gated secondary antibodies. B, quantitation of Syt1, Syt2, Syb1, and CaMexpression levels normalized to VCP in different brain regions. VCP was usedas internal loading control because it is ubiquitously expressed without nota-ble differences between cell types. The data shown are the means � S.E. **,p � 0.01; ***, p � 0.001 as analyzed by Student’s t test (n � 4 mice).

FIGURE 7. Ca2� binding to the C-lobe of CaM regulates Syt2 expression incortical neurons. Cultured cortical neurons from newborn wild-type or Syt1KO mice were infected at DIV 5 with control lentivirus, with CaM KD lentivirusexpressing CaM shRNAs without a CaM rescue construct, or with either wild-type CaM (� WT CaM) or mutant CaM in which Ca2� binding to the N-lobe(CaM1,2), the C-lobe (CaM3,4), or both lobes of CaM (CaM1,2,3,4) is blocked. Theneurons were analyzed at DIV 14 –15. A, schematic structures of wild-type andmutant CaMs. B, immunoblot analysis of the indicated proteins in control andCaM KD neurons expressing various CaM constructs. Note that to better indi-cate the expression level of Syt2, we did two different exposure times for theimmunoblots. The short exposure (short exp.) was �15 s, and the long expo-sure (long exp.) was �5 min. C, representative traces of IPSCs monitored inSyt1 KO neurons that were infected with the indicated lentiviruses. D, sum-mary graphs of the peak amplitudes of evoked IPSCs. The data shown are themeans � S.E. ***, p � 0.001 as analyzed by Student’s t test (n � number ofneurons indicated in bars from three independent cultures. E, kinetic analysisof the IPSC time course (n � 10 –17 in each group).

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endogenous ligands of neurexins and play important role insynapse formation (38–40). Our data indicate that a Ca2�-CaM paradigm might also play a role in synapse formation,although the functional consequences of these gene transcrip-tion changes remain to be further investigated. Third, the pos-sibly most important observation was that the CaM KD-in-duced changes in the expression of at least a subset of genescorrelates with the rostral-caudal expression pattern of thesegenes (Table 1). This correlation was extended to CaM itself, inthat our protein quantitations show that CaM is expressed at asignificantly higher level in forebrain, where expression of Syt2and Syb1 are suppressed, than in hindbrain, where they areactivated (Fig. 6). Thus, it is conceivable that CaMmay contrib-ute to the regulation of gene expression during development.CaM presumably acts via the Ca2�-dependent activation of

positive and negative transcription factors, whose identityremains unknown (Fig. 8). Our data demonstrate that Ca2�

binding to the C-lobe of CaM is both required and sufficient forits suppression of Syt2 expression (Fig. 7) and that CaM doesnot act via CaMKII� (supplemental Fig. S1). One possible path-way for CaM regulating Syt2 gene expression is through RE1silencing transcription factor (also known as the neuron-re-strictive silencing factor), because two RE1 locations have beenidentified on Syt2 gene (41). In addition, it has been shownthat HDAC4 and 5, which are CaMK-responsive repressorsof hypertrophic signaling, associate with neuron-restrictivesilencing factor and participate in neuron-restrictive silencingfactor-mediated repression of gene in ventricular myocytes(42). However, RE1 silencing transcription factor functions as ageneral silencer of neuron-specific genes (43, 44), and it ishighly unlikely that RE1 silencing transcription factor is nor-mally activated byCaM in cortical neurons to suppress Syt2 andSyb1 expression.In summary, our study shows that in cultured cortical neu-

rons, Ca2� binding to the C-lobe of CaM suppresses expressionof a subset of synaptic proteins that includes Syt2 and Syb1 andactivates expression of another subset of synaptic proteins thatincludes syntaxin-1A and Syt9. This regulation of gene expres-sion is developmental and is related to the normal rostral-cau-

dal expression pattern of the genes involved. Thus, CaMmedi-ates a Ca2�-dependent regulation of synaptic transmission thatgoes 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 Ca2�-binding mutant constructs and Kaishan Xian andIryna Huryeva for excellent technical assistance.

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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 (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 syntaxin-1 (Synt.) at DIV 14.

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

NM_130893 Scrt1 scratch homolog 1, zinc finger protein (Drosophila) 1.51 0.93 1.42 0.97

NM_008671 Nap1l2 nucleosome assembly protein 1-like 2 1.49 1.06 1.46 0.97

NM_009035 Rbpj recombination signal binding 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

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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

NM_001081557 Camta1 calmodulin binding transcription 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

NM_172659 Slc2a6 solute carrier family 2 (facilitated glucose transporter) 2.12 0.88 2.09 0.99

NM_054055 Slc13a3 solute carrier family 13 (sodium-dependent dicarboxylate 1.69 0.82 1.65 0.77

NM_153145 Abca8a ATP-binding cassette, sub-family A (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

NM_009199 Slc1a1 solute carrier family 1 (neuronal) 0.70 1.11 0.63 1.01

NM_011216 Ptpro protein tyrosine phosphatase, receptor type, O 0.70 1.01 0.57 1.15

NM_008983 Ptprk protein tyrosine phosphatase, receptor type, K 0.69 0.88 0.45 1.46

NM_011391 Slc16a7 solute carrier family 16 (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

NM_008746 Ntrk3 neurotrophic tyrosine kinase, 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

NM_009783 Cacna1g calcium channel, voltage-dependent, T type, alpha 1G 1.59 0.70 1.57 1.18

NM_011322 Scn1b sodium channel, voltage-gated, type I, beta 1.47 1.00 1.69 0.88

NM_133207 Kcnh7 potassium voltage-gated channel, subfamily H (eag-related) 0.71 1.14 0.54 1.36

NM_001099298 Scn2a1 sodium channel, voltage-gated, type II, alpha 1 0.71 1.03 0.60 1.30

NM_009782 Cacna1e calcium channel, voltage-dependent, R type, alpha 1E sub 0.68 0.93 0.61 1.25

NM_019697 Kcnd2 potassium voltage-gated channel, Shal-related family, member 2 0.66 0.94 0.54 0.95

NM_008170 Grin2a glutamate receptor, ionotropic, NMDA2A (epsilon 1) 0.65 1.01 0.45 1.00

NM_001034013 Accn1 amiloride-sensitive cation channel 1, neuronal 0.65 0.84 0.65 1.13

NM_153512 Kcng3 potassium voltage-gated channel, subfamily G, member 3 0.65 1.07 0.45 1.22

NM_007583 Cacng2 calcium channel, voltage-dependent, gamma subunit 2 0.61 0.95 0.69 0.98

NM_023872 Kcnq5 potassium voltage-gated channel, subfamily Q, member 5 0.60 1.02 0.42 0.99

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

NM_010601 Kcnh3 potassium voltage-gated channel, subfamily H (eag-related) 0.58 1.10 0.50 1.52

NM_001044308 Cacna1i calcium channel, voltage-dependent, alpha 1I subunit 0.46 0.99 0.47 1.04

NM_019430 Cacng3 calcium channel, voltage-dependent, gamma subunit 3 0.44 1.28 0.64 1.74

NM_026200 Kcnv1 potassium channel, subfamily V, 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

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

NM_016747 Dlg3 discs, large homolog 3 (Drosophila) 0.68 1.00 0.60 0.90

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

NM_198618 Dlgap3 discs, large (Drosophila) homolog-associated protein 3 0.67 1.20 0.69 1.17

NM_172910 Dlgap2 discs, large (Drosophila) homolog-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

NM_016695 Mpp2 membrane protein, palmitoylated 2 (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

NM_019518 Grasp GRP1 (general receptor for phosphoinositides 1)-associated 0.36 1.34 0.69 1.49

NM_018790 Arc activity regulated cytoskeletal-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

NM_178617 Necab1 N-terminal EF-hand calcium 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

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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

NM_175178 Aifm3 apoptosis-inducing factor, 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

NM_010717 Limk1 LIM-domain containing, protein kinase 1.54 0.90 1.57 0.67

ENSMUST00000055576 Pcsk6 proprotein convertase subtilisin 1.49 0.86 1.53 0.82

NM_172778 Maob monoamine oxidase B 1.48 0.93 1.76 1.02

NM_009035 Rbpj recombination signal binding protein for immunoglobulin kap 1.48 1.20 1.79 1.11

NM_029094 Pik3cb phosphatidylinositol 3-kinase, catalytic, beta polypeptid 1.46 0.72 2.07 1.04

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

NM_029933 Bcl9 B-cell CLL 0.71 0.85 0.62 0.88

NM_033264 Arpp21 cyclic AMP-regulated 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

NM_139147 Rab40b Rab40b, member RAS oncogene family 0.67 1.05 0.57 1.01

NM_177751 Cnksr2 connector enhancer of kinase 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

NM_021389 Sh3kbp1 SH3-domain kinase binding protein 1 0.64 1.24 0.62 0.94

NM_198114 Dagla diacylglycerol lipase, alpha 0.64 0.93 0.61 1.04

NM_080428 Fbxw7 F-box and WD-40 domain protein 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

NM_011201 Ptpn1 protein tyrosine phosphatase, non-receptor type 1 0.63 0.92 0.65 1.27

NM_001122998 Tiam2 T-cell lymphoma invasion and 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

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

NM_175930 Rapgef5 Rap guanine nucleotide exchange 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

NM_015743 Nr4a3 nuclear receptor subfamily 4, group A, member 3 0.58 1.17 0.68 1.10

NM_130862 Baiap2 brain-specific angiogenesis inhibitor 1-associated protei 0.56 0.85 0.60 1.20

NM_008926 Prkg2 protein kinase, cGMP-dependent, type II 0.48 1.35 0.63 1.35

NM_009528 Wnt7b wingless-related MMTV integration 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

NM_001080557 Vamp1 vesicle-associated membrane 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

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

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

NM_145219 Lgi3 leucine-rich repeat LGI family, member 3 1.87 0.63 2.36 0.77

NM_028880 Lrrtm1 leucine rich repeat transmembrane neuronal 1 0.71 0.96 0.66 0.98

NM_001080814 Fat3 FAT tumor suppressor homolog 3 (Drosophila) 0.70 1.19 0.58 1.35

NM_080285 Cttnbp2 cortactin binding protein 2 0.66 0.89 0.66 1.44

NM_020278 Lgi1 leucine-rich repeat LGI family, member 1 0.62 1.11 0.36 1.03

NM_008319 Icam5 intercellular adhesion molecule 5, 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

NM_178678 Lrrtm3 leucine rich repeat transmembrane 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

NM_172815 Rspo2 R-spondin 2 homolog (Xenopus 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

NM_021436 Tmeff1 transmembrane protein with EGF-like and two follistatin-l 0.71 0.99 0.69 1.11

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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

NM_001126047 Sema4c sema domain, immunoglobulin 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

NM_181074 Lingo1 leucine rich repeat and Ig domain containing 1 0.63 1.28 0.48 1.15

NM_013658 Sema4a sema domain, immunoglobulin 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

NM_009152 Sema3a sema domain, immunoglobulin 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

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

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

NM_011921 Aldh1a7 aldehyde dehydrogenase family 1, 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

NM_009780 C4b complement component 4B (Childo 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

NM_172604 Scara3 scavenger receptor class A, member 3 1.46 1.11 1.82 1.17

ENSMUST00000029046 Fabp5 fatty acid binding protein 5, 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

NM_011843 Mbc2 membrane bound C2 domain containing protein 1.44 0.96 2.69 1.06

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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

NM_011994 Abcd2 ATP-binding cassette, sub-family D (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

NM_173739 Galntl4

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-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

NM_013500 Hapln1 hyaluronan and proteoglycan link 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

BC021311 Zswim6 zinc finger, SWIM domain containing 6 0.69 1.06 0.69 0.98

NM_133706 Tmem97 transmembrane protein 97 0.69 1.59 0.67 1.47

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

NM_146073 Zdhhc14 zinc finger, DHHC domain 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

NM_153407 Csnrp2 cysteine-serine-rich nuclear protein 2 0.67 0.99 0.64 1.11

NM_198627 Vstm2l V-set and transmembrane domain 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

NM_028185 Lsm11 U7 snRNP-specific Sm-like protein 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

NM_033567 Cecr6 cat eye syndrome chromosome 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

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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

NM_145981 Phyhip phytanoyl-CoA hydroxylase interacting protein 0.59 0.87 0.41 0.98

NM_001039376 Pde4dip phosphodiesterase 4D interacting 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

NM_008255 Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 0.54 1.41 0.52 1.27

AK162044 March11 membrane-associated ring finger (C3HC4) 11 0.54 1.66 0.70 1.61

NM_019967 Dbc1 deleted in bladder cancer 1 (human) 0.54 1.31 0.68 1.41

NM_019675 Stmn4 stathmin-like 4 0.52 1.00 0.50 0.86

NM_201371 Prmt8 protein arginine N-methyltransferase 8 0.52 0.90 0.49 0.92

NM_198306 Galnt9

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylga 0.46 1.37 0.70 1.03

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

NM_001039167 D11Bwg0517e DNA segment, Chr 11, Brigham & 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

ENSMUST00000061282 OTTMUSG00000013918

predicted gene, 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

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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).

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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)

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.

1G Syt1 KO mIPSC Rise Time (ms)

Control 1.92 ± 0.11 18

CaM KD 1.83 ± 0.11 21 n.s.

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

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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

Syt2 (Control) 100 ± 39 3

Syt2 (CaM KD) 1045 ± 37 3 P<0.001

Syt2 (CaM KD+WT Res) 107 ± 41 3 n.s.

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

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

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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

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.

SCAMP (Control) 100 ± 32 3

SCAMP (CaM KD) 60 ± 16 3 n.s.

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

Spinal cord 1.05 ± 0.15 4 P<0.001

Syt2/VCP

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

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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