Supplementary Information Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

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1 Supplementary Information Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP Eunsil Cho, Dong-Hyun Kim, Young-Na Hur, Daniel J. Whitcomb, Philip Regan, Jung-Hwa Hong, Hanna Kim, Young Ho Suh, Kwangwook Cho, and Mikyoung Park 1

2 Supplementary Text Supplementary Result To test whether neurogenesis occurs during glycine-induced LTP, which is the preparation used in the present study, we performed Bromodeoxyuridine (BrdU) assay to mark newly dividing cells. As a control, we examined HEK293T cells that are labeled with BrdU antibodies after treatment with BrdU for 2 hours (data not shown) and 12 hours (Supplementary Figure 4), indicating that the cells undergo cell division. Expectedly, we found that neurons are not immunolabeled with BrdU antibodies during the treatment of BrdU for 12 hours at basal state and also during glycine-induced LTP (Supplementary Figure 4). Taken together, these results indicate neurons do not undergo cell division during glycine-induced LTP in cultured hippocampal neuronal system. Supplementary Method BrdU assay HEK293T cells or cultured hippocampal neurons were incubated with 10 µg/ml BrdU (Sigma) for 12 hr. BrdU (10 µg/ml) was included in all steps untill fixation. To detect the incorporated BrdU, cells were acid-washed to separate DNA into single strands with 1 N HCl (Sigma) for 10 min on ice and consecutively with 2 N HCl for 10 min at room temperature after fixation and permeation. Then, cells were incubated with anti-brdu antibody (1:100, Sigma) for 2 hr at room temperature. Cells were washed and incubated with Cy3-conjugated secondary antibody (1:300, Thermo scientific) for 1 hr at room temperature prior to fluorescent confocal microscopy. 2

3 CTX HC OB ST TH CB Supplementary Figure 1. In situ hybridization of ccny mrna in mouse brain. Image from the Allen Brain Atlas, OB, olfactory bulb; CTX, cortex; ST, striatum; HC, hippocampus; TH, thalamus; CB, cerebellum. Scale bar, 1000 m. 3

4 Ave intensity of CCNY (normalized) a Lenti- Ctrl #1 #2 #3 #4 CCNY GFP - tubulin 37 kda 25 kda 50 kda b psuper #2 Scrambled +Rescue CCNY-HA CCNY-HA CCNY-HA CCNY-HA c ** ## n=17 n=17 n=17 n=11 Supplementary Figure 2. Characterization of shrnas of CCNY. (a) All of four different shrnas for efficient knockdown of CCNY in cultured neurons. Note the similar expression level of GFP among the samples, indicating similar infection efficiency by each lentivirus. (b) CCNY-HA plasmid was co-transfected either with psuper-gfp control, psuper-gfp- #2, psuper-gfp-scrambled, or psuper-gfp- #2 plus CCNY shrna-resistant rescue CCNY-WT plasmid. CCNY-HA expression was almost completely blocked by CCNY shrna (), and this blockade was significantly rescued back to the control level (+Rescue). Scale bar, 20 μm. (c) Average intensity of CCNY-HA was analyzed in each sample. **p < relative to psuper-gfp control, ## p < relative to, student s t test. Data represent means ± SEM. 4

5 SEP-GluA1 (min:sec) 00:00 04:16 07:01 14:18 17:49 23:35 27:36 01:00 03:45 08:02 17:34 20:04 23:05 29:37 01:43 04:19 06:33 13:05 17:22 22:38 28:08 01:30 04:46 07:46 11:02 17:19 22:35 28:51 02:30 04:12 07:57 14:58 20:06 24:02 28:39 00:14 04:30 07:31 14:48 19:19 24:20 29:52 02:30 04:15 07:46 14:49 17:34 22:38 28:26 01:00 04:46 08:02 15:19 18:50 23:06 28:56 low Control CCNY-WT + Rescue high Supplementary Figure 3. More representative images of SEP-GluA1 before and after glycine stimulation. Pseudocolor intensity scale bar is shown. Scale bars, 1 μm each. 5

6 a BrdU (10 µg/µl) Pre-incubation with BrdU for 12 hr 3 min 0 hr 12 hr 25 min 15 min imaged imaged imaged imaged b Basal 0 hr 12 hr 25 min 40 min BrdU BrdU BrdU BrdU Cultured hippocampal neurons BrdU BrdU HEK293T Supplementary Figure 4. Neurogenesis does not occur during glycine-induced LTP. (a) Diagram of experimental scheme. Note that BrdU (10 µg/µl) is included in all steps during the entire experimental procedure. (b) No newly born neurons are observed during glycineinduced LTP in cultured hippocampal neurons. Note that HEK293T cells undergo cell division. Cell division was visualized by BrdU immunostaining. The white dotted shapes for cell bodies of neurons (upper images) or whole cell morphology of HEK293T cells (lower image). Scale bars, 20 μm. 6

7 Scrambled Scrambled Ctrl CCNY-WT Ctrl CCNY-WT DIV5 DIV11 DIV17 DIV24 DIV30 LS1 LP1 LS2 LP2 SPM Tx-100-soluble PSD DG CA3 CA1 CTX ST HC TH SN CB Fig. 1c, CCNY Fig. 1c, -tubulin Fig. 1d Fig. 1e, CCNY Fig. 1e, GluA1 Fig. 1e, -tubulin P0 P7 P14 P21P28 CCNY Prox1 Ctip2 Py -tubulin Fig. 1f, CCNY Fig. 1f, GluA1 Fig. 1f, -tubulin Fig. 1g, CCNY Fig. 1g, GluA1 H S1 P1 S2 P2 S3 P3 Fig. 1g, PSD-95 Fig. 1g, Synaptophysin Fig. 2h, CCNY Fig. 2h, GluA1 Fig. 2h, Fig. 2h, GluN1 GFP Fig. 2h, β-tubulin Fig. 4e, CCNY Fig. 4e, GFP Fig. 4e, β-tubulin Basal CCNY GluA1 GluN1 GFP β-tubulin p845-glua1 GluA1 CCNY GFP β-tubulin Basal Supplementary Fig. 2a, CCNY Supplementary Fig. 2a, GFP Supplementary Fig. 2a, -tubulin Supplementary Figure 5. Original blots for immunoblot analysis. 7

8 Supplementary Movie Legends Supplementary Movie 1. Overexpression of CCNY inhibits glycine-induced increase of SEP- GluA1 fluorescence. Time-lapse SEP-GluA1 images from spines of a hippocampal neuron coexpressing mcherry and SEP-GluA1 (Control; left panel) or CCNY-WT-mCherry and SEP- GluA1 (CCNY-WT; right panel). cine (200 M) stimulation is indicated by the letters. Time is indicated in min:sec. Supplementary Movie 2. Knockdown of CCNY facilitates glycine-induced increase of SEP- GluA1 fluorescence. Time-lapse SEP-GluA1 images from spines of a hippocampal neuron coexpressing CCNY shrna-mcherry and SEP-GluA1 (; left panel) or CCNY-rescuemCherry and SEP-GluA1 (+Rescue; right panel). cine (200 M) stimulation is indicated by the letters. Time is indicated in min:sec. 8