Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Figure S1. Localization of CtBP1 and time course of its neuronal activitycontrolled nuclear shuttling A. Localization of endogenous CtBP1 in synapses and nuclei in primary neuronal cultures. Neurons were counterstained for Bassoon or Homer as pre- and postsynaptic markers; nuclei were stained with DAPI. B. Cortical neurons with basal activity (control) and at various time-points upon pharmacological treatment to enhance neuronal activity. C. Cortical neurons with basal activity or upon block of neuronal activity. D. LB effect on CtBP1 nuclear shuttling under basal activity of the culture. E-G. Plots show quantification of the treatments shown in A-C on nuclear IF of endogenous CtBP1. Data information: In all graphs columns show means, whiskers indicate SEM, the numbers in the brackets indicate the number of quantified cells from at least two neuronal cultures; one-way ANOVA with Bonferroni posttest was used for statistics: ns– p>0.05,***– p<0.001.Scale bars are 10 µm on the overview image in A, B, C and D and 5 µm on the close up image in (A). Figure S2. Long-term imaging does not result in artificial rise in nuclear FPAEGFP A. Images show no spontaneous PA of CtBP1intPAEGFP during 3h of imaging before PA with 405nm laser pulse. The scale indicates false color coding of F PAEGFP intensity. Scale bar is 10 µm. B. Quantification of the fluorescence changes of CtBP1intPAEGFP in the ROI depicted in (A). Figure S3. CtBP1 controls expression of activity-regulated genes in neurons A. Structure of BDNFpI+II EGFP reporter: promoters I and II (black lines), exons I and II (boxes), cAMP response element (CRE) and RE1 are depicted. B. Nuclear IF intensities of CtBP1 are plotted against BDNFpI+II-driven expression of EGFP for individual cells and show negative correlation. C-D. Expression of BDNFpI+IIEGFP reporter in primary cortical neurons with basal vs. enhanced (C) or silenced (D) activity. Cells were counterstained with CtBP1 antibody and DAPI. E, F. Quantification of the nuclear FEGFP in experiments shown in C and D. G. Cultured cortical neurons were over-infected with lentiviruses expressing shRNAs 467, 944 or scrambled on the day of plating and harvested at DIV 16. Expression of synapsin, synaptophysin and CtBP1 was quantified in cell lysates, GAPDH was always used as a loading control. Synapsin 1,2 and synaptophysin were probed on the same membrane. H. Quantification of the effects shown in G. Expression of shRNAs 467 and 944, but not of scrambled shRNA caused a substantial reduction in the expression levels of CtBP1 (467: 4±1.2% of control; 944: 2.5±0.7% of control), while having no significant effect on the expression levels of two other neuronal proteins tested. I. mRNA expression of 14 activity-regulated genes is regulated after expression of shRNA467. Normalized mRNA levels relative to levels in neurons expressing scrambled shRNA are plotted. shRNA 467 yields comparable results as independent shRNA 944 (Fig 5A), what argues strongly against off-target effects of the shRNAs. Data information: In the graphs the numbers in the brackets indicate the number of analyzed cells (E, F) and loadings (H) from two independent experiments or qPCR reactions derived from samples from three independent cultures (I). Student’s t-test (E, F and I) or one-way ANOVA with Bonferroni posttest (H) was used for statistics: ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001. Scale bars are 10µm in C and D. Figure S4. CtBP1 is slightly reduced at synapses from Bsn-/- mice and is further depleted in Bsn-/-/Pclo KD neurons. A, B, C. Synaptic CtBP1 was stained in cultures in which equal numbers of neurons from WT and Bsn-/- neurons were mixed and plated (A) or in cultures infected with lentivirus expressing shRNA Pclo28 (B) or scrambled shRNA (C) together with EGFP-synapsin. Synapses were marked with staining for synaptophysin (A) or Homer (B,C). In A, synapses from Bsn-/- neurons lack Bassoon specific staining (arrowhead), which is clearly visible in synapses of WT neurons (arrows). In B and C synapses of transduced neurons express EGFP-synapsin (arrowheads). Synapses of neurons expressing Pclo28 shRNA show clear reduction in IF for CtBP1 (arrowhead in B) in comparison to synapses of non-transduced neurons (arrows in B). Synapses of neurons expressing scrambled shRNA (arrowheads in C) show no difference in the IF for CtBP1 when compared to synapses of non-transduced neurons (arrows in C). D, E. Quantification of synaptic CtBP1 IF in the experiment shown in A, B and C . F. Immunoblots of expression levels of CtBP1 in total brain lysates of newborn WT, Bsn-/-, Pclo3 and DKO mutant mice. G. Quantifications of CtBP1 signal ID in brain lysates from WT, Bsn -/-, Pclo3 and DKO mutant mice. H. Images showing the decrease of synaptic CtBP1 IF at the synapses of Bsn -/-/Pclo KD (Pclo 28) neurons. Synapses were marked by staining for synapsin. I. Quantification of the synaptic CtBP1 IF in Bsn-/-/Pclo28 neurons. Data information: Numbers in the brackets on the graphs represent the number of quantified synapses (D,E) or visual fields (I) from two neuronal cultures and independent loadings (G). Student’s t-test (D , E and I) or one-way ANOVA with Bonferroni posttest (G) were used for statistics; ns p>0.05, *** p<0.001. For all images: Colored images show overlay, color code is given in description, scale bars are 5µm. Figure S5. 4AP bicu enhances the neuronal network activity in neurons deficient for Bassoon and Piccolo A. Syt1 Ab uptake was used to evaluate the neuronal activity in WT/scrambled and Bsn-/-/Pclo28 neurons upon 4AP bicu stimulation. Cells were stained after fixation for synapsin to label synapses. Colored images show overlay. B. Quantification of the effect shown in A. Data information: Numbers in the brackets in the graph represent the number of quantified visual fields from three independent neuronal cultures. One-way ANOVA with Bonferroni posttest was used for statistics; ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001. Scale bar is 5µm. Figure S6. 2DG does not modulate the nuclear and the synaptic CtBP1levels upon activity block A. Overexpressed CtBP1intEGFP NAD/H bidning mutant displays aberrant synaptic localization. Cells were stained with Homer antibody to mark synapses. B. Enhanced neuronal activity reduces NAD/NADH ratio. Quantification of NAD/NADH ratio in primary cortical cultures with elevated synaptic activity (4AP bicu-2.5mM 50µM, 8h) and upon silencing (APV CNQX-50µM 10µM, 48h). Values are normalized to NAD/NADH ratio in cultures with basal activity (dashed line). C, D. Quantification of nuclear and synaptic CtBP1 IF from the experiments shown in E and F. E, F. Staining of CtBP1 in nuclei (E) and in synapses (F) of neurons in cultures with basal or blocked neuronal activity and with or without 2DG treatment. Synaptophysin staining was used to label synapses, DAPI to stain nuclei. G. Example images showing the propidium iodide (PI) staining to identify dead cells in control cultures and cultures treated with 5mM 2DG for 48h. Cells fixed with PFA were used as a positive control. Nuclei are stained with DAPI. White arrows indicate PI positive cells. H. Quantification of the effects shown in F. Data information: In the graphs, the numbers in the brackets indicate the number of of analyzed samples (B), cells (C) or visual fields (D, H). B shows data of one of two independent experiments, which yielded comparable results. B,C and H show pooled data derived from three independent neuronal cultures. One-way ANOVA with Bonferroni posttest was used for statistics: ns p>0.05, ** p<0.01, *** p<0.001. Colored images show overlays. Scale bars are 10µm in the overview image in A and in E, 5µm in the close up in A and in F and 50µm in G. Supplementary Table S1. Role of CtBP1 and its nuclear shuttling in the regulation of expression of activity-regulated genes Table summarizes results of qPCR analysis of expression of activity-regulated genes in control neurons infected with scrambled and shRNA944. Neurons with basal activity (control), with enhanced activity and with nuclear exit block were analyzed. Data information: The data is derived from 6 (scr) or 8 (shRNA944) qPCR reactions from 2 independent cultures and is normalized to the expression levels of the genes in neurons with basal activity for each infection. All values are shown as mean ± SEM. One-way ANOVA with Bonferroni posttest was used for statistics. The full gene names are listed.