Supplementary Information

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1 Supplementary Information Selective control of inhibitory synapse development by Slitrk3-PTPδ trans-synaptic interaction Hideto Takahashi 1, Kei-ichi Katayama 2, Kazuhiro Sohya 3,4, Hiroyuki Miyamoto 4,5, Tuhina Prasad 1, Yoshifumi Matsumoto 2, Maya Ota 2, Hiroki Yasuda 6, Tadaharu Tsumoto 3, Jun Aruga 2 and Ann Marie Craig 1 1. Brain Research Centre and Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada 2. Laboratory for Behavioral and Developmental Disorders, RIKEN Brain Science Institute (BSI), Wako-shi, Japan 3. Laboratory for Cortical Circuit Plasticity, RIKEN BSI, Wako-shi, Japan 4. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Saitama , Japan 5. Laboratory for Neurobiology of Synapse, RIKEN BSI, Wako-shi, Japan 6. Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Japan 1

2 CONTENTS Supplementary Figure 1....p. 3 Supplementary Figure 2....p. 5 Supplementary Figure 3....p. 6 Supplementary Figure 4....p. 7 Supplementary Figure 5....p. 9 Supplementary Figure 6....p. 10 Supplementary Figure 7....p. 11 Supplementary Figure 8....p. 12 Supplementary Figure 9....p. 14 Supplementary Figure p. 15 Supplementary Figure p. 16 2

3 Supplementary Figure 1. Analysis of surface expression, axon adhesion, GAD65 clustering and the uptake of synaptotagmin luminal domain antibody in fibroblast-neuron coculture for Slitrk isoforms. (a) Quantification of the average intensity of surface HA for the indicated HA-tagged protein-expressing COS cells. All HA-tagged constructs except HA-Slitrk5 and HA-Slitrk6 generated similar surface expression. Although HA-Slitrk5 and HA-Slitrk6 generated a lower surface expression level than HA-neuroligin2 (HA-NLG2), both had synaptogenic activity (Fig. 1d). Kruskal-Wallis ANOVA, P < 3

4 0.0001, n 60 cells each; *P < compared with HA-NLG2 and with HA-Slitrk2 by Dunn's pairwise post-hoc test. (b) Quantification of axonal contact area per 1,000 μm 2 of COS cell area for the indicated HA-tagged protein-expressing COS cells. Axonal contact area was higher for all Slitrk isoforms compared to negative control HA-CD4 (Kruskal-Wallis ANOVA, P < , n 60 cells each; *P < 0.001, #P < 0.01 compared with HA-CD4 by Dunn's pairwise post-hoc test), suggesting an effect on axon adhesion as well as presynaptic differentiation. (c) COS cells expressing HA-Slitrk3 (upper panel) but not HA-CD4 (lower panel) induced clustering of presynaptic GABA-producing enzyme GAD65 along contacting axons. (d) Quantification of the total integrated intensity of GAD65 staining associated with COS cells expressing the indicated HA-tagged proteins and not associated with gephyrin clusters divided by COS cell area. GAD65 clusters associated with the GABAergic postsynaptic scaffold gephyrin were presumed to correspond to endogenous synapses and thus excluded. Mann-Whitney's U test, *P < , n = 30 cells each. (e) COS cells expressing HA-Slitrk3 (upper panel) but not HA-CD4 (lower panel) induced functional presynaptic differentiation assessed by incubating live neurons with antibodies to synaptotagmin (SynTag) luminal domain. Quantification is shown in Figure 1f. Data are presented as mean ± s.e.m. Scale bars represent 20 μm. 4

5 Supplementary Figure 2. Low-level expression of YFP-Slitrk3 does not affect inhibitory or excitatory synapse number. We compared the densities of the indicated synaptic markers along dendrites between neurons transfected with YFP-Slitrk3-expressing vectors (Trans) and neighboring untransfected neurons (Untrans). There was no significant change in any synaptic marker including apposed VGAT/gephyrin clusters marking inhibitory synapses and apposed VGLUT1/PSD-95 clusters marking excitatory synapses. Data are presented as mean ± s.e.m. Mann-Whitney's U test, n = 24 neurons for each condition. 5

6 Supplementary Figure 3. Recombinant Slitrk3 localizes to the somatodendritic domain and not to axons. Cultured hippocampal neurons were co-transfected at 14 DIV with YFP-Slitrk3-expressing and mcherry-expressing vectors, and immunostained with MAP2 a marker for dendrites at 15 DIV. Axons of transfected neurons can be detected as mcherry-positive and MAP2-negative thin protrusions (arrowheads). Recombinant YFP-Slitrk3 expressed at low level in cultured hippocampal neurons was distributed and highly punctate in dendrites but hardly detected in axons (arrowheads). Scale bar represents 40 μm. 6

7 Supplementary Figure 4. Characterization of Slitrk3 shrna constructs. (a) Knockdown of Slitrk3 in heterologous cells by shrna. HEK cells were co-transfected with the indicated shrna vectors plus the indicated CFP-tagged Slitrk constructs for 24 h. We used pll(cmv)mcherry as the shrna vector, driving shrna from the U6 promoter and mcherry from the CMV promoter. Slitrk3*-CFP bears silent mutations rendering it RNAi-resistant against only 7

8 sh-slitrk3#1. Scale bar represents 20 μm. (b) Quantification of knockdown efficiency and specificity of shrna. sh-slitrk3#1 and sh-slitrk3#2 significantly knocked down the expression of Slitrk3-CFP and had no effects on the expression of Slitrk2-CFP and Slitrk1-CFP. The expression of Slitrk3*-CFP was knocked down by sh-slitrk3#1 but not by sh-slitrk3#2. Kruskal-Wallis ANOVA, P < , n > 20 images each (approximately transfected cells in a single image); *P < compared with sh-con by Dunn's pairwise post-hoc test. (c) Knockdown of endogenous Slitrk3 in cultured hippocampal neurons by shrna. Cultured hippocampal neurons at 0 DIV were transfected with the indicated shrna vectors by Amaxa nucleofection at plating and grown at high density directly on polylysine-coated culture dishes in Neurobasal media with B27 supplement (Invitrogen), followed by immunoblotting at 14 DIV with the anti-slitrk3 antibody (Santa Cruz Biotechnology) and anti-β-actin antibody (Abcam, ab8227). The Slitrk3 band around 135 kda was reduced by sh-slitrk3#1 and sh-slitk3#2. (d) Densitometric analysis of Slitrk3 protein expression in hippocampal cultures transfected by Amaxa nucleofection. Data were normalized by β-actin expression level. Kruskal-Wallis ANOVA, P < 0.01, n = 6 culture experiments, *P < 0.01 compared with sh-con by Dunn's pairwise post-hoc test. (e) Quantification of the estimated Slitrk3 protein expression level of only transfected cells based on the Amaxa transfection efficiency of 58.0 ± 2.5% (n = 6 Amaxa transfections). Data were normalized by β-actin expression level. Kruskal-Wallis ANOVA, P < 0.01, n = 6 culture experiments, *P < 0.01 compared with sh-con by Dunn's pairwise post-hoc test. Data are presented as mean ± s.e.m. 8

9 Supplementary Figure 5. Slitrk3-deficient mice have normal gross brain morphology. Cresyl violet stain of brain sagittal sections showed no gross morphological defects between wild-type (Slitrk3 +/+ ) and Slitrk3 / mice. Scale bar represents 2 mm. 9

10 Supplementary Figure 6. Ca ++ -independent interaction between Slitrk3 and PTPδ. (a,b) Binding of recombinant Slitrk3-Fc proteins to COS cells expressing HA-PTPδ did not require extracellular calcium, as measured by bound Slitrk3-Fc per surface HA-PTPδ in extracellular solution (ECS) or in Ca ++ free ECS containing 10 mm EGTA. The average intensity of bound Slitrk3-Fc protein was normalized by the surface HA expression level of HA-PTPδ on COS cells because of the decrease in surface expression of HA-PTPδ in Ca ++ -free conditions. Data are presented as mean ± s.e.m. Mann-Whitney's U test, P = 0.85, n > 20 cells each. 10

11 Supplementary Figure 7. PTPδ-Fc protein binds to Slitrk3 and also to all other Slitrk isoforms. COS cells expressing extracellular HA-tagged Slitrk1 6, Neuroligin2 (NLG2) or CD4 were incubated with recombinant PTPδ-Fc proteins. Bound PTPδ-Fc (upper images) and surface HA (lower images) were co-immunostained without permeabilization for imaging analysis. All HA-tagged proteins showed significant surface expression on COS cells. Recombinant PTPδ-Fc proteins bound to COS cells expressing Slitrk1 6 but not NLG2 or negative control CD4. Quantitative analysis is shown in Figure 7d. Scale bar represents 20 μm. 11

12 Supplementary Figure 8. Trans-synaptic coclustering of axonal PTPδ and dendritic or somatic Slitrk3 at inhibitory synaptic contact sites. 12

13 (a) HA-PTPδ expressed in GABAergic axons (Upper left) and YFP-Slitrk3 expressed in the target neuron (Upper right) were accumulated with each other at contact sites (identified by HA-PTPδ and MAP2, middle right) between the axons and the dendrites (arrows) or the cell soma (arrowheads) of the target neuron. VGAT was also accumulated at those contact sites (Bottom). (b) High magnification images showing the axon-dendrite contact sites (Upper) and the axon-soma contact sites (Bottom) in (a). Scale bars represent 20 μm (a) and 5 μm (b). 13

14 Supplementary Figure 9. shrna with a scrambled sequence for sh-ptpδ has no effect on VGAT accumulation induced by Slitrk3 in coculture. (a) Cultured hippocampal neurons were transfected at 0 DIV by Amaxa nucleofection with a vector co-expressing ECFP and either control shrna (sh-con), scrambled shrna (sh-scb) or shrna against PTPδ (sh-ptpδ). The transfected neurons were cocultured at 14 DIV with COS cells expressing Slitrk3-YFP and immunostained for VGAT at 15 DIV. A majority of axons were transfected with shrna-expressing vectors. COS cells expressing Slitrk3 induced VGAT clustering along contacting axons of sh-scb-transfected neurons to about the same level as contacting axons of sh-con-transfected neurons. Slitrk3 induced little VGAT clustering along contacting axons of PTPδ knockdown neurons compared to those of neurons transfected with sh-con or sh-scb. (b) Total integrated intensity of VGAT staining on CFP-positive axons associated with COS cells expressing Slitrk3 and not associated with MAP2 (images not shown) divided by the CFP-positive axon contact area. Kruskal-Wallis ANOVA, P < , n = 30 cells for each condition; *P < compared with sh-con or sh-scb by Dunn's pairwise post-hoc test. n.s., not significant (P > 0.05). The data are representative of three independent experiments different from the population data for Figure 8. Data are presented as mean ± s.e.m. Scale bar represents 20 μm. 14

15 Supplementary Figure 10. Slitrk2 induces the clustering of axonal PTPδ with VGAT by trans-interaction. (a) COS cells expressing Slitrk2-CFP induced YFP-PTPδ accumulation on contacting axons with VGAT. Neurons were co-transfected with YFP-PTPδ and mcherry and then cocultured with COS cells expressing Slitrk2-CFP. Transfected axons were detected as mcherry-positive and MAP2-negative processes (Upper Left). (b) Total integrated intensity of YFP-PTPδ associated with COS cells expressing Amigo-CFP or Slitrk2-CFP and not associated with MAP2 divided by mcherry-positive axon contact area. Mann-Whitney's U test, *P < , n = 18 for Amigo-CFP and Slitrk2-CFP. Data are presented as mean ± s.e.m. Scale bars represent 10 μm (a) and 2 μm (inset). 15

16 Supplementary Figure 11. Slitrk2 requires PTPδ for induction of inhibitory, but not excitatory, presynaptic differentiation. (a,c) Cultured hippocampal neurons were transfected at 0 DIV by Amaxa nucleofection with a vector co-expressing ECFP and either control shrna (sh-con), scrambled shrna (sh-scb) or shrna against PTPδ (sh-ptpδ). The transfected neurons were cocultured at 14 DIV with COS cells expressing Slitrk2-YFP and immunostained for VGAT (a) or VGLUT1 (c) at 15 DIV. A majority of axons were transfected with shrna-expressing vectors. COS cells expressing Slitrk2 induced little VGAT 16

17 clustering along contacting axons of PTPδ knockdown neurons compared to contacting axons of control neurons transfected with sh-con or sh-scb (a). In contrast, COS cells expressing Slitrk2 induced VGLUT1 clustering along contacting axons of PTPδ knockdown neurons as well as those of control neurons (c). (b,d) Total integrated intensity of VGAT staining (b) and VGLUT1 staining (d) on CFP-positive axons associated with COS cells expressing Slitrk2 and not associated with MAP2 (images not shown) divided by the CFP-positive axon contact area. Kruskal-Wallis ANOVA, P < , n = 30 cells for each condition; *P < compared with sh-con or sh-scb by Dunn's pairwise post-hoc test. n.s., not significant (P > 0.05). Data are presented as mean ± s.e.m. Scale bars represent 20 μm. 17