Disrupted-in-Schizophrenia-1 (DISC1) regulates spines of the glutamate synapse via Rac1

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1 Disrupted-in-Schizophrenia-1 () regulates spines of the glutamate synapse via Rac1 By Akiko Hayashi-Takagi, Manabu Takaki, Nick Graziane, Saurav Seshadri, Hannah Murdoch, Allan J Dunlop, Yuichi Makino, Anupamaa J Seshadri, Koko Ishizuka, Deepak P. Srivastava, Zhong Xie, Jay M. Baraban, Miles D. Houslay, Toshifumi Tomoda, Nicholas J. Brandon, Atsushi Kamiya, Zhen Yan, Peter Penzes, and Akira Sawa Nature Neuroscience: doi:1.138/nn.2487

2 S S Brains P1 P2 sucrose density gradient synaptosomal fraction.5% TritonX-1 S1.5% TritonX-1 PSD1.5% Sarcosyl Synaptophysin Rat whole brain 1-Triton 2-Triton Sarcosyl S1 PSD1 S2 PSD2 S3 PSD3 Total Synaptosome S2 PSD2 S3 PSD3 b. 1 DIV 7 DIV 3 DIV -tubulin -tubulin Merge Merge Merge Supplementary Figure 1: Localization of in the dendritic spine of mature neurons. (a) Enrichment of in the postsynaptic density. Left, PSD fractionation protocol. Right, Western blotting of each synaptic fraction with antibodies against (D27), synaptophysin (pre-synaptic marker), and (post-synaptic marker). Full-length (arrowhead) is concentrated in the fractions of the postsynaptic density (PSD1 and PSD2). (b) Distribution change of during neuronal maturation. While localization of is prominent at the centrosome in developing primary cortical neuron (arrowhead) (1 day in vitro: 1 DIV), postsynaptic, which co-localizes with, becomes prominent in the mature neuron (3 DIV). Scale bar, 1 μm. Nature Neuroscience: doi:1.138/nn.2487

3 Virus (Primary neurons) No treatment Empty virus RNAi #2 RNAi #1 No treatment Empty virus RNAi #2 64 RNAi #1 64 RNAi #1 GluR1 RNAi GFP/MAP2, N-terminal -tubulin 5, D tubulin Kal-12 Kal-9 Kalirin 25 b. Plasmid (293 Cells) -HA β-tubulin Mock Cont RNAi RNAi # Days after transfection 5 (%) knockdown (%) Days after transfection Plasmid-based RNAi Lentivirus-based RNAi c. Plasmid (293 Cells) d. -HA β-tubulin RNAi #1 -FL -FL R - - R 5 Virus (Primary neurons) (%) OAS1 induction relative to no infection 2 1 n.s. RNAi #1 Supplementary Figure 2: Effects of two independent RNAi against. (a) Successful knockdown of endogenous by lentivirus-based RNAi against in primary cortical neurons. According to published studies 25, we used shrnas to two independent target sequences of (RNAi #1 and #2), or scrambled sequence of RNAi #1 (). Left: majority of neurons (stained by MAP2 antibody in magenta) were infected with lentiviral vector-mediated shrna indicated in green. Scale bar, 2 μm. Middle: Two independent antibodies from different groups (D27 and N-terminal) were used to test the effects of RNAi (#1 and #2 targeted for exon 1 and 6, respectively). isoforms around 1 kda detected by N-terminal antibody (antigen, exons 2-6) are almost totally abolished by both RNAi (red asterisks), whereas some isoform(s) around 1 kda detected by D27 (antigen, amino acids in exon 11) is knocked down only with RNAi#1, but not with RNAi#2 (blue asterisks). β-tubulin, internal control. Right: No influence on the expression of GluR1,, and Kalirin by RNAi#1. (b) knockdown by RNAi in the time course. Left, representative blot of knockdown for -HA in HEK293 cells. Right, the effects by two vector systems (plasmid-based or lentivirus-based) are shown. Plasmid-based RNAi markedly knocks down from 1 day after transfection, whereas lentivirus-based RNAi (used for biochemical studies such as those of Figs. 3b and 5a) takes 4 days for knockdown after infection. (c) Validation of rescue constructs (-FL R or DISC- R ) that contains 3 nucleotide mutations without alteration of deduced amino acids. FL, full length; DISC-, mutant lacking binding domain of. -FL R and DISC- R are resistant against RNAi (asterisks), while -FL and - are effectively knocked down by RNAi (double asterisks). (d) Quantitative real-time PCR for OAS1. No difference in expression level of OAS1 was observed between cells with control RNAi and those with RNAi #1. Nature Neuroscience: doi:1.138/nn.2487

4 RNAi #1 GFP merge GFP merge b. GFP merge RNAi #1 GFP merge Supplementary Figure 3: Knockdown of in dendritic spines by RNAi. (a) Knockdown of exogenously expressed -HA in spines by RNAi #1. Synaptic -HA was examined by immunofluorescent cell staining with an antibody against HA. (b) Knockdown of endogenous in spines by RNAi #1. The primary cortical neuron at 24 DIV were transfected with RNAi #1 or control RNAi, followed by immunocytochemistry with use of antibody 2 days after transfection. Nature Neuroscience: doi:1.138/nn.2487

5 Size (μm 2 ) RNAi # Trasfected plasmid (μg) Spine density/1 μm Trasfected plasmid (μg) Supplementary Figure 4: Dose effects of RNAi on spines morphology. Cortical neurons (1.5 x 1 5 cells/4 cm 2 ) were transfected with.5, 1, or 2 μg of control RNAi or RNAi #1 at 21 DIV, followed by analyses 2 days after transfection. Dose difference influenced the extent of spine size and density modestly, although transfection efficiency was dramatically affected (data not shown). P <.5, P <.1. Nature Neuroscience: doi:1.138/nn.2487

6 Surface GluR1 intensity per area (A.U.) # Co-localization with synaptophysin (%) 1 5 RNAi #1 Supplementary Figure 5: Increase in surface expression of GluR1 upon knockdown. Increases in the surface expression of the AMPA-type glutamate receptor GluR1, a key mechanism of synaptic potentiation, was observed in the dendritic spine of rat primary cortical neuron in the presence of RNAi #1. Ratio of spine that was co-localised with synaptophysin immunoreactivity was not altered upon knockdown. #P <.1. Nature Neuroscience: doi:1.138/nn.2487

7 constructs c. full-length Y2H b. 1 constructs 1, AA , AA , AA , AA (35-394) full-length peptide array GST GST-Kal7 DKAETLQQRLEDLEQEKISLHFQLP LQQRLEDLEQEKISLHFQLPSRQPA EDLEQEKISLHFQLPSRQPALSSFL EKISLHFQLPSRQPALSSFLGHLAA Interaction with Interaction with d. e. IP: Y2H HEK293 IB: PDE4B Mock -FL -FL + PDE4B - + PDE4B binding PDE4B binding IP: IP: Input IP: Input PC12 IB: KIF5 (endo) IB: IB: Mock -FL - full-length full-length GAPDH 62 (35-394): = Input PDE4B Input KIF5 (endo) Supplementary Figure 6: Domain mapping of for binding with. (a) Amino acids of are crucial for binding with. Various deletion constructs of HA-tagged were co-transfected with full-length in HEK293 cells. Binding of with was examined by co-immunoprecipitation. IP, antibodies against these target molecules were used for co-immunoprecipitation. IB, antibodies against these target molecules were used for Western blotting. (b) Domain mapping of for interaction. Various deletion constructs were used for assays. Y2H indicates the portion of identified in yeast two-hybrid screening with a bait of fragment (amino acid 1-382). (c) peptide array showing that amino acids of are crucial for binding. An array of immobilized peptide spots of overlapping 25-mer peptides, each shifted along by 5 amino acids in the entire sequence of full-length (-FL), was probed with -GST (amino acid ) or GST alone and detected by immunoblotting with antibody against. Schematic of, which indicates binding site for or PDE4B, is shown. (d) binding site of (amino acids ) is not crucial for interaction of and PDE4B. Deletion of binding site (Δ) does not affect binding of with PDE4B in co-immunoprecipitation. (e) binding site of (amino acids ) is not crucial for interaction of and endogenous (endo) KIF5. Deletion of binding site (Δ) does not affect binding of with KIF5 in co-immunoprecipitation. Nature Neuroscience: doi:1.138/nn.2487

8 -FL - Venus Venus Venus Venus Supplementary Figure 7: inhibits -induced spine enlargement. was co-transfected with either full-length (-FL) or mutant that lack binding domain (-Δ) into primary cortical neurons. To delineate the spine morphology, GFP was also co-transfected. Both -FL and -Δ were co-localized with mainly in the dendritic spines. inhibits -induced spine enlargement, but -Δ does not. Nature Neuroscience: doi:1.138/nn.2487

9 GFP endo GFP/ endo RNAi #1 GFP endo GFP/ endo Supplementary Figure 8: No influence of RNAi to localization to the spine. No influence of knockdown on the localization of endogenous (endo ) in the spines (arrowheads). Nature Neuroscience: doi:1.138/nn.2487

10 / binding (%) RNAi #1 b. / binding (%) NR1/ binding (%) / binding (%) n.s / binding (%) Sham ECT c. / binding (%) / binding (%) Sham APV withdrawal Supplementary Figure 9: Protein interaction of // influenced by activation of the NMDA-type glutamate receptor. (a) Decrease in / binding upon lentivirus-based knockdown in the rat primary culture. (b) Decrease in interactions among,, and three min after electroconvulsive treatment (ECT) in rat brains in vivo. (c) Decrease in interactions of / and / in the rat primary neuron culture after selective activation of the NMDA receptor by APV withdrawal (WD). P <.5. Nature Neuroscience: doi:1.138/nn.2487

11 FL-MBP Total lysate Soluble fraction After purification b ( )-GST 188 Total lysate Soluble fraction After purification c. -FL-GST 25 Total lysate Soluble fraction After purification 15 1 Supplementary Figure 1: Preparation of recombinant,, and. The expressed fusion proteins [-FL-MBP (a), -( )-GST (b), and -FL-GST (c)] in soluble fraction from E. Coli were purified by glutathione or amylose beads. Recombinant proteins were subjected with centrifugal filter devices to concentrate protein solution and to remove some undesirable products less than 1 kda (After purification). Each fraction of purification procedure was subjected to SDS-PAGE, and coomassie brilliant blue staining was performed. Nature Neuroscience: doi:1.138/nn.2487

12 Recombinant (pmol) Cortical lysate (protein/μg) 5 1 IP: 25 5 IPed reconbinant DISC (A.U.) Endogenous in cortical lysate (1 μg of protein) corresponds to.34 pmol of recombinat 2 1 Y = 84.4x R2 = Purified recombinant (/pmol) Preciputated endogenous (A.U.) 2 1 Y =.38x R2 = Cortical lysate (Protein/μg) b. + (pmol) Cortical lysate (protein/μg) 5 1 co-ip: 25 5 co-iped reconbinant (A.U.) pmol of co-iped with 2. pmol of in cortical lysate (1 μg of protein) Y = 24.6x R2 = Purified recombinant (/pmol) Preciputated endogenous (A.U.) 2 1 Y =.27x R2 = Cortical lysate (Protein/μg) c. + (pmol) co-ip: Cortical lysate (protein/μg) 25 5 co-ip reconbinant (A.U.) pmol of co-iped with 1.9 pmol of in cortical lysate (1 μg of protein) Y = 35.1x R2 = Purified recombinant (/pmol) Preciputated endogenous (A.U.) 2 1 Y =.35x R2 = Cortical lysate (Protein/μg) Supplementary Figure 11: Stoichiometric analysis of // binding. (a) Amount of protein immunoprecipitated (IPed) with an anti- antibody mex3 from cortical lysate was estimated by immunosignal in Western blotting, compared to that of IPed recombinant protein (-FL-MBP). Signal from.34 pmol of recombinant corresponds to that from endogenous in 1 μg of cortex protein lysates. (b, c) Immunoreactivity of recombinant -GST or -GST co-immunoprecipitated (co-iped) with mex3 from the mixture of -GST ( pmol) or -GST ( pmol) together with -MBP (.34 pmol, estimated amount of endogenous in 1 μg of cortex lysates) was compared to immunoreactivity of endogenous or from immunoprecipiates of cortical lysates with the same antibody. Through these experiments, the amounts of and co-iped with in cortical lysates were estimated. Nature Neuroscience: doi:1.138/nn.2487

13 (%) 25 GEF activity (A. U.) Rac1 plus vehicle Rac1 plus b (%) 25 (sec) GEF activity (A. U.) 2 15 Cdc42 plus vehicle Cdc42 plus 1 c (sec) (%) GEF activity (A. U.) 2 15 RhoA plus vehicle RhoA plus (sec) Supplementary Figure 12: In vitro GEF activity of for Rac1, Cdc42, and RhoA. Recombinant GST--( ) influenced mant-gtp binding to His-Rac1, but not His-Cdc42, nor His-RhoA. For assessment of GEF activity of GST- in vitro, RhoGEF exchange assay biochem kit (Cytoskeleton Inc, CO, USA) was used, according to the manufacture s instructions. Briefly, the fluorescent signal at 46 nm from mant-gtp bound to RhoGTPase (Rac1, Cdc42, or RhoA) was measured with a fluorescence plate leader, Fluoskan ascent FL (Thermoscientific, Vantaa, Finland), in the presence or absence of GST-. Nature Neuroscience: doi:1.138/nn.2487

14 Co-transfection with -Myc -Myc Rac1-DN-HA Mock Rac1-DN RNAi RNAi 36 β-tubulin 5 b. Spine density/1 μm 8 4 Mock Rac1 -DN Co-transfection Spine density (%) Rac1-DN relative to Mock RNAi #1 RNAi #2 Supplementary Figure 13: Effect of dominant negative form of Rac1 (Rac1-DN) on -mediated spine morphology. (a) No difference in expression level of Rac1-DN in the presence or absence of RNAi (asterisks). (b) Blockade of RNAi-induced increase in spine density by expression of Rac1-DN. 1.5 μg of psuper-venus RNAi were co-transfected with 1.5 μg of prk-rac1-dn into the primary cortical neuron at the density of 1.5 x 1 5 cells on the 18 mm dish. Significant effects of Rac1-DN compared to mock are shown as P <.5, P <.1. Nature Neuroscience: doi:1.138/nn.2487

15 (%) (%) Signal upon RNAi relative to control RNAi expression Rac1 activity / binding Days after infection b. IB: Input IP Cont RNAi RNAi #1 RNAi #1 Days after infection Supplementary Figure 14: Increased Rac1 activity and decreased / binding by knockdown in the time course. (a) Time course changes of knockdown effect and correlative parameters. Lentivirus-based RNAi #1 elicits knockdown of 4 days after infection into cortical neurons. Corresponding to this change, increased Rac1 activity and decreased / binding are observed. (b) Representative immunoblot showing the / binding was decreased upon knockdown (asterisk). Nature Neuroscience: doi:1.138/nn.2487

16 Size (μm 2 ) # 2 Length (μm) 1 Breadth (μm) # Spine density/1 μm 8 4 RNAi #1 Supplementary Figure 15: Long-term suppression of leads to spine shrinkage in slices and brains in vivo. Effect of long-term knockdown on spine morphology in rat organotypic cortical culture. P <.5, #P <.1. Nature Neuroscience: doi:1.138/nn.2487

17 2+ Ca AMPAR NMDAR AMPAR AMPAR NMDAR 2+ Ca dissociation Pak1 Rac1 GDP NMDAR activation Rac1 GDP Rac1 Pak1 GTP P spine enlargement Summary of experiments and detected phenotypes Transfection Short-term Long-term RNAi -FL overexpression - overexpression Rac1-CA Rac1-DN Bigger Smaller no change Bigger Smaller Smaller Smaller no change Smaller Smaller Supplementary Figure 16: Summary table and figure. We propose a model that inhibits from accessing Rac1 at baseline by anchoring in the protein complex of //, whereas activation of NMDA-type glutamate receptor results in dissociation of this protein complex, allowing access of to Rac1 and proper spine enlargement. The table summarizes influence of on spine morphology in contrast to that of Rac1 (also see Figs. 1, 2, 6, and 7). Effect of RNAi resembles that of Rac1-CA (constitutive-active), whereas the impact of -FL is similar to that of Rac1-DN (dominant-negative). Short-term, 2 days after transfection of plasmid-based constructs. Long-term, 6 days after transfection of plasmid-based constructs Nature Neuroscience: doi:1.138/nn.2487

18 For Fig 2 a. Input Myc IP IP IP Input Myc Input Myc Input Whole Synapto IP: Whole Myc IP: Synapto Myc Kal Kalilirin 25 Tiam1 βpix For Fig 3 a. b. IP IP 25 Cont RNAi RNAi #1 EGFP -FL - Supplementary Fig. 17 Full immunoblot of key experiments (Figs 2 and 3). The cropped areas shown in the main figures are indicated by dashed line. Nature Neuroscience: doi:1.138/nn.2487

19 For Fig 4 IP IP a. b. Sham ECT 25 Sham ECT 25 IP Basal APV WD 25 NR1 For Fig 5 a. b. RNAi #1 RNAi #2 EGFP -FL - GTP-Rac1 17 IP Rac1 Rac1 Rac1 Total Rac1 17 Rac1 17 Phospho -Pak1 62 EGFP -FL - Total Pak1 62 Supplementary Fig. 18 Full immunoblot of key experiments (Figs 4 and 5). The cropped areas shown in the main figures are indicated by dashed line. Nature Neuroscience: doi:1.138/nn.2487