Fig 1: Knockdown of Pde10a ameliorates the abnormal behaviors associated with the partial loss of miR-137.a, Schemati illustration of the sh-Pde10a lentivirus constructs. Lentivirus encoding shRNA targeting Pde10a (sh-Pde10a) or negative control (sh-Neg) were injected into adult miR-137flox/+ and miR-137flox/+;Nestin-Cre mice 3 weeks before the behavioral assays.b, The ICC staining by using PDE10A antibody revealed a high knockdown efficiency of sh-Pde10a. The experiment was repeated independently three time with similar results.c, In the Morris water maze test, the mean escape latency for the trained mice decreased over the course of the 5 learning days in all groups, and sh-Pde10a resulted in the improved latency to locate the platform in miR-137flox/+;Nestin-Cre mice (Day5: miR-137flox/+ + sh-Pde10a vs. miR-137flox/+;Nestin-Cre + sh-Pde10a: t = 2.738, P = 0.2172; miR-137flox/+ + sh-Neg vs. miR-137flox/+;Nestin-Cre + sh-Neg: t = 5.873, P = 0.0003; Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; ***, P < 0.001). In the spatial probe test performed on day 6, although sh-Pde10a did not change the swimming speed (F3,33 = 0.2683, P = 0.8478), it significantly ameliorated the impaired latency to the platform (F3,33 = 5.377, P = 0.0040) and increased the number of target crossings (F3,33 = 3.073, P = 0.0411) in miR-137flox/+;Nestin-Cre mice. Data represent means ± s.e.m; miR-137flox/++sh-Neg: n = 9 mice; miR-137flox/++sh-Pde10a: n = 10 mice; miR-137flox/+;Nestin-Cre+sh-Neg: n = 9 mice; miR-137flox/+;Nestin-Cre+sh-Pde10a: n = 9 mice. One-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; *, P < 0.05.d, In the three-chamber test, sh-Pde10a had no significant effect on the preference to the left or right chamber of miR-137flox/+ and miR-137flox/+;Nestin-Cre mice during the habituation phase. Left vs. right: miR-137flox/++sh-Neg mice (n = 9 mice), t = 1.446, P > 0.9999; miR-137flox/++sh-Pde10a mice (n = 10 mice), t = 2.464, P > 0.9999; miR-137flox/+;Nestin-Cre+sh-Neg mice (n = 9 mice), t = 2.079, P > 0.9999; miR-137flox/+;Nestin-Cre+sh-Pde10a mice (n = 9 mice), t = 0.2697, P > 0.9999. In the subsequent task probing phase, the application of sh-Pde10a ameliorated the impaired sociability and social novelty in miR-137flox/+;Nestin-Cre mice, as indicated by the significantly increased interacting time with a mouse versus empty cage (miR-137flox/+;Nestin-Cre+sh-Pde10a mice: tEmpty cage vs. Stranger1 = 6.742, P < 0.0001) or with novel mice (Stranger 2) versus familiar mice (Stranger 1) (miR-137flox/+;Nestin-Cre+sh-Pde10a mice: tStranger2 vs. Stranger1 = 9.225, P < 0.0001). Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; ***, P < 0.001; ****, P < 0.0001.e-h, sh-Pde10a could rescue the impaired neuronal phenotype in vitro. (e) Primary hippocampal neurons were isolated from P0 littermate of from miR-137flox/+ and miR-137flox/+;Nestin-Cre mice (n = 3 mice). After infecting lentivirus encoding sh-Pde10a, we confirmed its efficiency with GFP and immunocytochemistry staining of MAP2 were performed at DIV 7. The experiment was repeated independently three time with similar results. (f) sh-Pde10a reduced dendritic complexity in miR-137flox/+;Nestin-Cre neurons compared with controls, as determined by Sholl analysis (Data represent means ± s.e.m; Interaction: F57,1340 = 3.286, P < 0.0001; Distance: F19,1340 = 795.4, P < 0.0001; Treatment: F3,1340 = 177.5, P < 0.0001. Two-way ANOVA with Bonferroni post hoc test.). sh-Pde10a significantly reduced the dendritic length (miR-137flox/+;Nestin-Cre mice: tsh-Neg vs. sh-Pde10a = 2.822, P = 0.0354) (g) and the number of dendritic nodes (miR-137flox/+;Nestin-Cre mice: tsh-Neg vs. sh-Pde10a = 3.485, P = 0.0046) (h) in miR-137flox/+; Nestin-Cre neurons. n = 23 slice from 3 mice per group. Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., n onsignificant; *, P < 0.05; **, P < 0.01.
Fig 2: PDE10A is a key mRNA target of miR-137.a, Primary screen of miR-137 predicted targets in HEK293FT cells. 3’UTR-dependent luciferase assays were performed using both sh-control (miR-137NC), and sh-miR-137 (miR-137OE) for each of 10 miR-137 predicted targets. For each 3’UTR, luciferase expression was normalized (hRluc/hluc) to the miR-137NC control treatment. 8 out of 10 predicted targets were significantly regulated by miR-137 in vitro. n = 3 independent experiments; Data represent means ± s.e.m; tEmpty vector = 1.951, P = 0.1227; tPtpn2 = 5.854, P = 0.0042; tPde10a = 6.736, P = 0.0025; tSatb2 = 12.79, P = 0.0002; tEpha7 = 15.58, P = 0.0009; tCttnbp2nl = 3.631, P = 0.0221; tFoxp1 = 4.305, P = 0.0126; tFam3c = 9.047, P = 0.0008; tTsn = 3.890, P = 0.0177; Unpaired Two-Tailed t-test; n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.b–c, MiR-137 regulates the expression of Pde10a through the predicted binding site in the 3’UTR of Pde10a. (b) The miR-137 7mer-1A target site in the Pde10a-3’UTR as predicted by TargetScan was removed. (c) Co-transfection experiment was performed for the four plasmids, including “miR-137NC (miR-137 negative control)”, “miR-137OE (miR-137 overexpression)”, “Pde10a-3’UTR” and “Pde10a-3’UTR?miR-137” constructs in HEK293FT cells (combination as indicated in the figure). All the other three samples were normalized to “miR-137NC + Pde10a-3’UTR”. There is no significant difference when directly comparing Pde10a-3’UTR and Pde10a-3’UTR?miR-137 constructs. Pde10a-3’UTR-dependent expression of a luciferase reporter gene was suppressed by miR-137 overexpression. MiR-137-mediated suppression of luciferase was specific, as deletion of the miR-137 target site in the Pde10a-3’UTR (Pde10a-3’UTR?miR-137) abolished repression by miR-137 overexpression. n = 9 independent experiments. Data represent means ± s.e.m; miR-137NC + Pde10a vs. miR-137OE + Pde10a: t = 4.511, P = 0.0009; miR-137NC + Pde10a vs. miR-137 NC + Pde10a?miR-137: t = 1.22, P > 0.9999; miR-137OE + Pde10a vs. miR-137OE + Pde10a?miR-137: t = 8.373, P < 0.0001; miR-137 NC + Pde10a?miR-137 vs. miR-137OE + Pde10a?miR-137: t = 2.642, P = 0.0856; Two-way ANOVA with Bonferroni post hoc test; n.s., nonsignificant; ***, P < 0.001; ****, P < 0.0001.d, Partial loss of miR-137 resulted in a significant increase in PDE10A (relative to ACTIN), which associated with the reduced phosphorylation of PKA (P-PKA / PKA-Ca) without changing total PKA levels (n = 3 mice). Full-length blots are presented in Supplementary Fig. 15. Data represent means ± s.e.m; tPDE10A = 3.92, P = 0.0172; tp-PKA = 12.12, P = 0.0003; tPKA-Ca = 0.691, P = 0.5274; tP-PKA/PKA-Ca = 8.059, P = 0.0013; Unpaired Two-Tailed t-test. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 3: Inhibition of PDE10A ameliorates the abnormal behaviors associated with the partial loss of miR-137.a, In the Mrris water maze test, the mean escape latency for the trained mice decreased over the course of the 4 learning days in all groups, but papaverine significantly improved the latency to locate the platform in miR-137flox/+;Nestin-Cre mice than in miR-137flox/+ mice (Day4: miR-137flox/+;Nestin-Cre + Vehicle vs. miR-137flox/+;Nestin-Cre + Papaverine: t = 2.831, P = 0.0321; miR-137flox/+ + Papaverine vs. miR-137flox/+;Nestin-Cre + Papaverine: t = 1.36, P > 0.9999; Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; *, P < 0.05). In the spatial probe test performed on day 5, although papaverine did not significantly change the swimming speed (F3,34 = 1.053, P = 0.8466), it ameliorated the impaired latency to the platform (F3,34 = 19.93, P < 0.0001) and increased the number of target crossings (F3,34 = 6.532, P = 0.0013) in miR-137flox/+;Nestin-Cre mice. Data represent means ± s.e.m; miR-137flox/++Vehicle: n = 9 mice; miR-137flox/++Papaverine: n = 9 mice; miR-137flox/+;Nestin-Cre+Vehicle: n = 10 mice; miR-137flox/+;Nestin-Cre+Papaverine: n = 10 mice. One-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; *, P < 0.05; **, P < 0.01 ****, P < 0.0001.b, In self-grooming test, papaverine resulted in significantly less time spent grooming in miR-137flox/+;Nestin-Cre mice (F3,59 = 5.96, P = 0.0013). Data represent means ± s.e.m; miR-137flox/++Vehicle: n = 16 mice; miR-137flox/++Papaverine: n = 16 mice; miR-137flox/+;Nestin-Cre+Vehicle: n = 16 mice; miR-137flox/+;Nestin-Cre+Papaverine: n = 15 mice. One-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; **, P < 0.01.c, In the marble-burying test, papaverine ameliorated the impaired repetitive behaviors in miR-137flox/+;Nestin-Cre mice demonstrating by the improved number of marbles buried to the same level in miR-137flox/+ mice (F3,62 = 14.01, P < 0.0001). Data represent means ± s.e.m; miR-137flox/++Vehicle: n = 17 mice; miR-137flox/++Papaverine: n = 16 mice; miR-137flox/+;Nestin-Cre+Vehicle: n = 17 mice; miR-137flox/+;Nestin-Cre+Papaverine: n = 16 mice. One-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; ***, P < 0.001; ****, P < 0.0001.d, In the three-chamber test, papaverine had no significant effect on the preference to the left or right chamber of miR-137flox/+ and miR-137flox/+;Nestin-Cre mice during the habituation phase. Left vs. right: miR-137flox/++Vehicle mice (n = 9 mice), t = 0.2517, P > 0.9999; miR-137flox/++Papaverine mice (n = 9 mice), t = 2.417, P > 0.9999; miR-137flox/+;Nestin-Cre+Vehicle mice (n = 10 mice), t = 1.397, P > 0.9999; miR-137flox/+;Nestin-Cre+Papaverine mice (n = 10 mice), t = 0.8727, P > 0.9999. In the subsequent task probing phase, the application of papaverine ameliorated the impaired sociability and social novelty in miR-137flox/+;Nestin-Cre mice, as indicated by the significantly increased interacting time with a mouse versus empty cage (miR-137flox/+;Nestin-Cre+Papaverine mice: tEmpty cage vs. Stranger1 = 3.705, P = 0.0119) or with novel mice (Stranger 2) versus familiar mice (Stranger 1) (miR-137flox/+;Nestin-Cre+Papaverine mice: tStranger2 vs. Stranger1 = 3.356, P = 0.0364). Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.e, Papaverine rescued the impaired long-term potentiation (LTP) in miR-137flox/+;Nestin-Cre mice. Left panel, A typical experiment showing the time course of CA1 LTP for a single recording. fEPSP traces before and after are shown in the inset above. Pooled data are showing the time course of LTP from all recordings made from miR-137flox/+ or miR-137flox/+;Nestin-Cre mice. Right panel, Average LTP amplitude measured at 55–60 min post-induction. n = 6 slices from 4 mice per group; 10 fEPSP slope (%) values were collected from 1 slice. Data represent means ± s.e.m; t = 0.078, P = 0.9379; Unpaired Two-Tailed t-test; n.s., nonsignificant.f, Increased paired-pulse facilitation (PPF) in papaverine-treated miR-137flox/+;Nestin-Cre mice. PPF studies across different interpulse intervals (20 ms, 50 ms, 100 ms, 200 ms and 400 ms) revealed that papaverine could restore the decreased paired-pulse ratio in miR-137flox/+;Nestin-Cre mice. Left panel, Representative recording of the paired-pulse ratio at the interpulse interval of 50 ms from the slices prepared from papaverine-treated miR-137flox/+ and miR-137flox/+;Nestin-Cre mice. Left panel, Paired-pulse ratio at an interval of 50 ms measured for up to 40 events for each recording in papaverine-treated miR-137flox/+ and miR-137flox/+;Nestin-Cre mice. n = 8 slice from 4 mice per group; Data represent means ± s.e.m; t = 0.2954, P = 0.7720; Unpaired Two-Tailed t-test; n.s., nonsignificant.g, Partial loss of miR-137 resulted in a significant increase in PDE10A (relative to ß-Actin), which associated with the reduced phosphorylation of PKA (P-PKA / PKA-Ca) without changing total PKA levels. After injecting papaverine, the reduced phosphorylation of PKA in miR-137flox/+;Nestin-Cre mice has resorted to the same level in miR-137flox/+ mice (n = 3 mice). Full-length blots are presented in Supplementary Fig. 15. Data represent means ± s.e.m; One-way ANOVA with Bonferroni post hoc test. Pde10a: F3,8 = 11.66, P = 0.0027; P-PKA: F3,8 = 6.085, P = 0.0184; PKA-Ca: F3,8 = 0.2374, P = 0.8679; P-PKA/PKA-Ca: F3,8 = 24.66, P = 0.0002. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Supplier Page from MilliporeSigma for Anti-PDE10A (C-terminal) antibody produced in rabbit