Fig 1: SPIN90 is necessary for PSD95 dissociation during cLTD. (A) DIV18–21 WT, Spin90-KO and AAV-6xMyc-SPIN90 infected Spin90-KO neurons (SPIN90Res) hippocampal neurons expressing GFP and PSD95 were pre-incubated with CNQX (10 µM) for 1 h before cLTD treatment, followed by immunostaining with GFP (green) and PSD95 (red) antibody. Scale bars indicate 5 µm. (B) The localization of PSD95 in ratios of PSD95 to GFP puncta was analyzed (n = 13–21 for WT and Spin90-KO; n = 19 for SPIN90Res-control(Con), n = 21 for SPIN90Res-LTD). (C) DIV18 WT, Spin90-KO and SPIN90Res neurons were subjected to cLTD treatment followed by harvesting of cells and immunoblot analysis. Phosphorylation of PSD95 at Thr19 was assessed. (D) Quantification of pThr19-PSD95 levels normalized to total PSD95 after cLTD induction (n = 9 for WT and Spin90-KO; n = 3 for SPIN90Res). All data are expressed as mean ± SEM (#p = 0.0629, *p < 0.05, **p < 0.01, n.s., non-significant).
Fig 2: Schematics of SPIN90 during NMDAR-LTD. (A) LTD induction in normal (WT) neurons; (1) Upon NMDAR stimulation, SPIN90 binds to Akt, sequestering it from phosphorylating downstream GSK3ß. (2) Active GSK3ß is able to phosphorylate PSD95 at Thr19, which causes (3) PSD95 to dissociate from GluA2-containing AMPAR, which subsequently induces AMPAR internalization and LTD. (B) LTD induction in Spin90-KO neurons; (1) Absence of SPIN90 allows activated Akt (phosphorylation at Ser473 sequesters GSK3ß, thereby (2) hindering GSK3ß from phosphorylating PSD95. (3) GluA2-containing AMPARs remain “docked” on PSD95, unable to internalize. The defect in AMPAR internalization and LTD in Spin90-KO neurons may consequently have secluded excess AMPARs on the surface, which is apparent in Figures 3A–C.
Fig 3: Expression of glutamate receptor 2 (GluR2) and glycogen synthase kinase 3ß (GSK-3ß) during postnatal development and in Shank3b-/- ACC. (A) Western-blotting of GluR2 in the ACC of 1, 2, 3, 4 and 6 weeks (w) old mice. (B) Western-blotting of p-GSK-3ß (S9) in the ACC of 1, 2, 3, 4 and 6 w old mice. Notice the increase of GluR2 and decrease of p-GSK-3ß (S9) from 3 w on. (C) Western-blotting of GluR2, p-GSK-3ß, total GSK-3ß (t-GSK-3ß), phosphorylated PSD-95 (pPSD-95), phosphorylated Akt (p-Akt), and total Akt (t-Akt) in WT and Shank3b-/- ACC. Notice the up-regulation of p-GSK-3ß and p-Akt, and down-regulation of GluR2 and pPSD-95 in Shank3b-/- ACC. Values represent mean ± SEM. *P < 0.05. **P < 0.01. N = 6 mice per group. Student’s t-test.
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