Partial loss of psychiatric risk gene Mir137 in mice causes repetitive behavior and impairs sociability and learning via increased Pde10a

Abstract

Genetic analyses have linked microRNA-137 (MIR137) to neuropsychiatric disorders, including schizophrenia and autism spectrum disorder. miR-137 plays important roles in neurogenesis and neuronal maturation, but the impact of miR-137 loss-of-function in vivo remains unclear. Here we show the complete loss of miR-137 in the mouse germline knockout or nervous system knockout (cKO) leads to postnatal lethality, while heterozygous germline knockout and cKO mice remain viable. Partial loss of miR-137 in heterozygous cKO mice results in dysregulated synaptic plasticity, repetitive behavior, and impaired learning and social behavior. Transcriptomic and proteomic analyses revealed that the miR-137 mRNA target, phosphodiesterase 10a (Pde10a), is elevated in heterozygous knockout mice. Treatment with the Pde10a inhibitor papaverine or knockdown of Pde10a ameliorates the deficits observed in the heterozygous cKO mice. Collectively, our results suggest that MIR137 plays essential roles in postnatal neurodevelopment and that dysregulation of miR-137 potentially contributes to neuropsychiatric disorders in humans.

Figure 1:. Loss of miR-137 leads to postnatal lethality.

a, Generation of the miR-137 conditional allele. A targeting vector was designed to disrupt the Mir137 gene via homologous recombination in mouse embryonic stem cells, where two loxP sites were inserted upstream (~2 kb) and downstream (~0.6 kb) of the Mir137 gene (the image was modified from the UCSC Genome Browser). b, Schematic of the crosses to generate miR-137 knockout mice. Green arrows indicate the primer sets that designed for PCR genotyping. By crossing with either Zp3-Cre or Nestin-Cre line, we could specifically delete Mir137 in the germline or central and peripheral nervous system and generate the heterozygous global knockout (miR-137^+/–) and conditional knockout (miR-137^flox/+;Nestin-Cre) mice. c–d, Gross appearance of miR-137 gKO mice. Representative wild-type (miR-137^+/+), heterozygous (miR-137^+/–) and homozygous (miR-137^–/–) of miR-137 knockout mice and brain tissues (c). At postnatal day 16 (P16), the body and brain weight of miR-137^–/– mice are much smaller than miR-137^+/+ and miR-137^+/– mice (n = 5 mice per group) (d). Data represent means ± s.e.m; Body: F_2,12 = 1123, P < 0.0001; Brain: F_2,12 = 67.05, P < 0.0001; One-way ANOVA with Bonferroni post hoc test; n.s., nonsignificant; ****, P < 0.0001. e, Verification of decreased expression of mature miR-137 in miR-137^flox/+;Nestin-Cre and miR-137^flox/flox;Nestin-Cre mice in the hippocampus using independent real-time PCR (n = 4 mice per group). Data represent means ± s.e.m; F_2,9 = 505.6, P < 0.0001; One-way ANOVA with Bonferroni post hoc test; ****, P < 0.0001. f, Kaplan-Meier graph shows survival curves of the miR-137^flox/+ (n = 13 mice), miR-137^flox/+;Nestin-Cre (n = 14 mice) and miR-137^flox/flox;Nestin-Cre (n = 11 mice) mice groups. Mice in the homozygous (miR-137^flox/flox;Nestin-Cre) group exhibited significantly decreased survival (dot) relative to the other groups (Two-Tailed log-rank tests; P < 0.0001). All 11 tested miR-137^flox/flox;Nestin-Cre mice died by postnatal day 28.

Figure 2:. Loss of miR-137 in the nervous system leads to synaptic overgrowth and impaired dendritic growth in vivo.

a, Immunohistochemistry (IHC) staining of PSD-95 and Synaptophysin in miR-137 cKO mice hippocampal tissues. PSD-95 and Synaptophysin IHC staining were carried out on 40-μm thick floating sections containing hippocampal tissue from four pairs of littermate cKO mice, i.e., miR-137^flox/+, miR-137^flox/+;Nestin-Cre and miR-137^flox/flox;Nestin-Cre mice, at the age of postnatal day 18 (P18) (n = 4 mice per group). Relative fluorescence intensity of PSD-95 (F_2,9 = 9.494, P = 0.0061) and Synaptophysin (F_2,9 = 12.73, P = 0.0024) elevated upon the loss of miR-137 in the hippocampus. Image analyses and quantification were performed using ImageJ software. Data represent means ± s.e.m; One-way ANOVA with Bonferroni post hoc test; n.s., nonsignificant; *, P < 0.05; **, P < 0.01. b–e, Golgi staining reveals dendritic growth in the hippocampal CA1 region in miR-137^flox/+ and miR-137^flox/+;Nestin-Cre mice (n = 13 slices from 4 mice per group). (b)The spine densities of basal (t = 3.895, P = 0.0007) and apical (t = 5.406, P < 0.0001) spines were significantly increased in the miR-137^flox/+;Nestin-Cre mice compared to the littermate control (Data represent means ± s.e.m. Unpaired Two-Tailed t-test; ***, P < 0.001; ****, P < 0.0001). (c) Sholl analysis showing, compared with neurons in miR-137^flox/+ mice, the CA1 pyramidal neurons in miR-137^flox/+;Nestin-Cre mice exhibited increased dendritic complexity (Data represent means ± s.e.m; Basal spines: t₈₀ = 3.952, P = 0.0016; t₉₀ = 4.534, P = 0.0001; t₁₁₀ = 3.047, P = 0.0437. Apical spines: t₇₀ = 3.210, P = 0.0388; t₁₁₀ = 4.105, P = 0.0013; t₁₂₀ = 4.004, P = 0.0019; t₁₃₀ = 5.242, P < 0.0001; t₁₄₀ = 3.493, P = 0.0143. Two-way ANOVA with Bonferroni post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; distance points with P < 0.05 were not marked in the figure). Partial loss of miR-137 significantly increased dendritic length (t_Basal = 3.275, P = 0.0023; t_Apical = 3.905, P = 0.0003; t_Total = 4.422, P < 0.0001) (d) and dendritic nodes (t_Basal = 2.118, P = 0.0377; t_Apical = 2.675, P = 0.0092) (e) in miR-137^flox/+;Nestin-Cre neurons (Data represent means ± s.e.m. Unpaired Two-Tailed t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3:. Partial loss of miR-137 leads to the learning and memory deficits.

a–d, MiR-137^flox/+;Nestin-Cre mice exhibited learning and memory deficits in the Morris water maze test. During the training phase, in which four trials were conducted per day for four successive days, both miR-137^flox/+ (n = 9 mice) and miR-137^flox/+;Nestin-Cre (n = 10 mice) mice showed improved latency to locate the platform, but miR-137^flox/+;Nestin-Cre mice exhibited a significant delay to diminish the latency to locate the platform. (Data represent means ± s.e.m; t_Day1 = 0.026, P > 0.9999; t_Day2 = 2.565, P = 0.0501; t _Day3 = 3.766, P = 0.0014; t_Day4 = 6.761, P < 0.0001; Two-way ANOVA with Bonferroni post hoc test. **, P < 0.01; ****, P < 0.0001) (a). In probe trials on Day 5, there was no significant difference in swimming speeds between the two groups of mice (t = 0.524, P = 0.6073) (b). Compare to miR-137^flox/+, miR-137^flox/+;Nestin-Cre mice exhibited a significantly longer latency to locate the platform (t = 5.530, P < 0.0001) (c), but less number of target crossings (t = 3.213, P = 0.0051) (d). Data shown are means ± s.e.m. Unpaired Two-Tailed t-test; **, P < 0.01; ****, P < 0.0001. e–h, The miR-137 cKO mice were subject to the Barnes maze test. During the training phase, in which four trials were conducted per day for four successive days, the miR-137^flox/+;Nestin-Cre mice (n = 15 mice) failed to diminish the latency of first entrance to the hiding box, indicating that loss of miR-137 results in impaired spatial memory (e). (Data represent means ± s.e.m; t_Day1 = 0.842, P > 0.9999; t_Day2 = 1.864, P = 0.2603; t _Day3 = 2.220, P = 0.1141; t_Day4 = 4.025, P = 0.0004; Two-way ANOVA with Bonferroni post hoc test. ***, P < 0.001. Probe trials on Day 5 demonstrated that miR-137^flox/+;Nestin-Cre mice also had spatial memory deficits as they spent less time (t = 2.537, P = 0.0173) in the target quadrant (f) and visited the target hole less often (t = 3.978, P = 0.0005) (g) than miR-137^flox/+ mice (n = 14 mice). The swimming speeds showed no significant difference between two groups (t = 1.713, P = 0.0981) (h). Data shown are means ± s.e.m. Unpaired Two-Tailed t-test; n.s., nonsignificant; *, P < 0.05; ***, P < 0.001. i–j, Partial loss of miR-137 resulted in impaired long-term potentiation (LTP) in miR-137^flox/+;Nestin-Cre mice. A typical experiment showing the time course of CA1 LTP for a single recording. fEPSP traces before (gray) and after (blue or red) are shown in the inset above. Pooled data showing the time course of LTP from all recordings made from miR-137^flox/+ or miR-137^flox/+;Nestin-Cre mice (i). Average LTP amplitude measured at 55–60 min post-induction (j). n = 8 slices from 4 mice per group; 10 fEPSP slope (%) values were collected from 1 slice. Data represent means ± s.e.m; t = 7.552, P < 0.0001; Unpaired Two-Tailed t-test; ****, P < 0.0001. k-l, Paired-pulse facilitation (PPF). Representative recording of the paired-pulse ratio at the interpulse interval of 50 ms from the slices prepared from miR-137^flox/+ (n = 7 slices from 4 mice) and miR-137^flox/+;Nestin-Cre (n = 6 slices from 4 mice) mice (k). PPF studies across different interpulse intervals (20 ms, 50 ms, 100 ms, 200 ms and 400 ms) revealed a significant difference in the paired-pulse ratio at 50 ms intervals (l). Data represent means ± s.e.m; t_20ms = 0.660, P = 0.5226; t_50ms = 3.389, P = 0.0060; t_100ms = 1.387, P = 0.2301; t_200ms = 0.905, P = 0.3893; t _400ms = 0.025, P = 0.9809. Unpaired Two-Tailed t-test; n.s., nonsignificant; **, P < 0.01.

Figure 4:. Partial loss of miR-137 causes impaired social behaviors in mice.

a, Self-grooming test showing an increased total time spent grooming (t = 2.410, P = 0.0257) and number of bouts (t = 2.939, P = 0.0081) in miR-137^flox/+;Nestin-Cre mice than in miR-137^flox/+ mice (n = 11 mice per group). Data represent means ± s.e.m; Unpaired Two-Tailed t-test; n.s., nonsignificant; *, P < 0.05, **, P < 0.01. b, In the marble-burying test, compared with miR-137^flox/+ mice (n = 11 mice), miR-137^flox/+;Nestin-Cre mice (n = 12 mice) showed a significant number of marble burying bouts. Data represent means ± s.e.m; t = 2.195, P = 0.0396; Unpaired Two-Tailed t-test; *, P < 0.05. c, In the open field test, miR-137^flox/+;Nestin-Cre mice (n = 12 mice) traveled similar distances (t = 0.7779, P = 0.4453) in 30-minutes duration and exhibited significantly less number of entries in the center zone (t = 2.292, P = 0.0323) when compared with control miR-137^flox/+ mice (n = 11 mice). Data represent means ± s.e.m; Unpaired Two-Tailed t-test; n.s., nonsignificant; *, P < 0.05. d, Social interactions tests are showing total time in contact and number of arena exploration. When comparing with miR-137^flox/+ mice (n = 10 mice), miR-137^flox/+;Nestin-Cre mice (n = 8 mice) displayed significantly less time in contact with unfamiliar mice (t = 2.326, P = 0.0335) and showed a similar arena exploration level (t = 1.320, P = 0.2055). Data represent means ± s.e.m; Unpaired Two-Tailed t-test; n.s., nonsignificant; *, P < 0.05. e–g, MiR-137^flox/+;Nestin-Cre mice exhibited impaired social behavior in the three-chamber test. Both miR-137^flox/+ (n =14 mice) and miR-137^flox/+;Nestin-Cre (n = 15 mice) mice had no preference for either left chamber or right chamber during the habituation phase (miR-137^flox/+ mice: t_{left vs. right} = 0.3572, P > 0.9999; miR-137^flox/+;Nestin-Cre mice: t_{left vs. right} = 1.545, P = 0.3785) (e), but miR-137^flox/+;Nestin-Cre mice lacked a social preference for a mouse over the empty cage (miR-137^flox/+ mice: t_{Empty vs. Stranger1} = 5.827, P < 0.0001; miR-137^flox/+;Nestin-Cre mice: t_{Empty vs. Stranger1} = 1.476, P = 0.8747; miR-137^flox/+ vs. miR-137^flox/+;Nestin-Cre: t_Stranger1 = 3.159, P = 0.0155) (f). Moreover, unlike miR-137^flox/+ control mice, miR-137^flox/+;Nestin-Cre mice displayed impaired social novelty recognition by demonstrating no preference for a novel mouse over a familiar mouse (miR-137^flox/+ mice: t_{Stranger2 vs. Stranger1} = 3.438, P = 0.0068; miR-137^flox/+;Nestin-Cre mice: t_{Stranger2 vs. Stranger1} = 0.3359, P > 0.9999; miR-137^flox/+ vs. miR-137^flox/+;Nestin-Cre: t_Stranger2 = 3.039, P = 0.0188) (g). Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; **, P < 0.01; ****, P < 0.0001. h-i, In the social discrimination test, miR-137^flox/+;Nestin-Cre mice exhibited impaired social discrimination ability. When comparing with miR-137^flox/+ mice (n = 13 mice), miR-137^flox/+;Nestin-Cre mice (n = 14 mice) exhibited significantly fewer entries number (miR-137^flox/+ mice: t_{Novel vs. Familiar} = 3.581, P = 0.0046; miR-137^flox/+;Nestin-Cre mice: t_{Novel vs. Familiar} = 0.722, P > 0.9999; miR-137^flox/+ vs. miR-137^flox/+;Nestin-Cre: t_Novel = 5.609, P < 0.0001) (h) and relatively lower % time spent in novel mouse zone (miR-137^flox/+ mice: t_{Novel vs. Familiar} = 1.538, P = 0.7822; miR-137^flox/+;Nestin-Cre mice: t_{Novel vs. Familiar} = 0.011, P > 0.9999; miR-137^flox/+ vs. miR-137^flox/+;Nestin-Cre: t_Novel = 1.689, P = 0.5846) (i). Data represent means ± s.e.m; Two-way ANOVA with Bonferroni post hoc test. n.s., nonsignificant; **, P < 0.01; ****, P < 0.0001. j–k, Partial loss of miR-137 resulted in imbalanced excitation/inhibition (E/I) ratio and impaired social behavior in miR-137 knockout mice. A representative whole-cell recording experiment (repeated independently three times with similar results) and samples trace at indicated time points showing the time course of evoked synaptic currents in the absence or presence of various inhibitors to determine the E/I ratio. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were pharmacologically isolated by using respective inhibitors specific to glutamate or GABA receptors. E/I ratio by sequentially recording evoked synaptic responses, firstly in the absence of any inhibitors to obtain total synaptic currents (i.e., EPSC+IPSC), secondly in the presence of NBQX/APV to obtain IPSC, and finally in the presence of NBQX/APV/Picrotoxin to verify the IPSC component. Sample traces are showing various components of synaptic currents (j). Summary data shows a decreased E/I ratio in miR-137^flox/+; Nestin-Cre (n = 8 slices from 4 mice) compared to miR-137^flox/+ control mice (n = 11 slices from 6 mice) (k). t = 2.311, P = 0.034; Unpaired Two-Tailed t-test. *, P < 0.05.

Figure 5:. Systematic identification of in vivo mRNA targets of miR-137 by integrating proteomic, transcriptomic and bioinformatic analyses.

a, Workflow of the TMT (Tandem Mass Tag)-based quantitative proteomic analysis. Six brain cortical samples from two litters (3 genotypes per little) were used as described in Method section. There were total ~1,800, 000 MS2 scans, among which ~140, 000 unique peptides and ~12,000 unique proteins were identified and quantified. b, SDS-PAGE and Coomassie staining of the whole brain cortical tissues homogenized in the lysis buffer before protein digestion for the proteomics analysis. c, The consistency of protein TMT quantification result was found between the two replicates. As a representative, miR-137^–/– samples in litter 1 (KO1) and litter 2 (KO2) mice showed high correlation regarding protein TMT quantification intensity. d, GO enrichment analysis of differentially expressed (DE) proteins (miR-137^–/– / miR-137^+/+) identified in the proteomic analysis (n = 150 for upregulated DE genes; n = 227 for downregulated DE genes). The DE genes were enriched in the biological process of “DNA replication,” “Nervous system development” and “Peptidase activity regulation.” The full result of the analysis is presented in Supplementary Fig. 8b. (e) Interactome of the differentially expressed (DE) genes upon the loss of miR-137. Predicted protein-protein interaction network, which was generated with represented upregulated genes (blue circle) and downregulated genes (red circle), indicates the impact of the miR-137 loss of function in various biological processes. Colored dots indicate the genes are differentially expressed in proteomics analysis. f, The overlap between miR-137 predicted targets and upregulated proteins. Proteomic analysis confidently identified 417 differentially expressed (DE) protein (377 genes) in miR-137^–/– mice compared to miR-137^+/+ mice. By overlapping with miR-137 predicted targets (1140 transcripts with conserved sites), we found that miR-137 predicted targets significantly overlapped with upregulated DE genes (DE_Up, 41 out of 150), but not down-regulated genes (DE_Down). Pearson’s chi-squared test calculated the p-value. g, MiR-137-mediated regulation in ASD candidate genes interactome. Predicted ASD-gene network exploring brain-specific interactions between ASD candidate genes were generated, including 15 upregulated and 16 downregulated DE proteins. Upregulated miR-137 predicted targets might potentially influence the adjacent ASD candidate gene’s expression in mRNA and protein levels, such as significantly downregulated GRIA3 and GABRB1 were previously reported as associated with ASD. The dots marked with bold lines indicate the ASD candidate genes targeted by miR-137 as shown in Supplementary Fig. 9.

Figure 6:. 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-137_NC), and sh-miR-137 (miR-137_OE) for each of 10 miR-137 predicted targets. For each 3’UTR, luciferase expression was normalized (hRluc/hluc) to the miR-137_NC 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; t_{Empty vector} = 1.951, P = 0.1227; t_Ptpn2 = 5.854, P = 0.0042; t_Pde10a = 6.736, P = 0.0025; t_Satb2 = 12.79, P = 0.0002; t_Epha7 = 15.58, P = 0.0009; t_Cttnbp2nl = 3.631, P = 0.0221; t_Foxp1 = 4.305, P = 0.0126; t_Fam3c = 9.047, P = 0.0008; t_Tsn = 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-137_NC (miR-137 negative control)”, “miR-137_OE (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-137_NC + 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-137_NC + Pde10a vs. miR-137_OE + Pde10a: t = 4.511, P = 0.0009; miR-137_NC + Pde10a vs. miR-137 _NC + Pde10a^∆miR−137: t = 1.22, P > 0.9999; miR-137_OE + Pde10a vs. miR-137_OE + Pde10a^∆miR−137: t = 8.373, P < 0.0001; miR-137 _NC + Pde10a^∆miR−137 vs. miR-137_OE + 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-Cα) without changing total PKA levels (n = 3 mice). Full-length blots are presented in Supplementary Fig. 15. Data represent means ± s.e.m; t_PDE10A = 3.92, P = 0.0172; t_p-PKA = 12.12, P = 0.0003; t_PKA-Cα = 0.691, P = 0.5274; t_{P-PKA/PKA-Cα} = 8.059, P = 0.0013; Unpaired Two-Tailed t-test. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7:. 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-137^flox/+;Nestin-Cre mice than in miR-137^flox/+ mice (Day4: miR-137^flox/+;Nestin-Cre + Vehicle vs. miR-137^flox/+;Nestin-Cre + Papaverine: t = 2.831, P = 0.0321; miR-137^flox/+ + Papaverine vs. miR-137^flox/+;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 (F_3,34 = 1.053, P = 0.8466), it ameliorated the impaired latency to the platform (F_3,34 = 19.93, P < 0.0001) and increased the number of target crossings (F_3,34 = 6.532, P = 0.0013) in miR-137^flox/+;Nestin-Cre mice. Data represent means ± s.e.m; miR-137^flox/++Vehicle: n = 9 mice; miR-137^flox/++Papaverine: n = 9 mice; miR-137^flox/+;Nestin-Cre+Vehicle: n = 10 mice; miR-137^flox/+;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-137^flox/+;Nestin-Cre mice (F_3,59 = 5.96, P = 0.0013). Data represent means ± s.e.m; miR-137^flox/++Vehicle: n = 16 mice; miR-137^flox/++Papaverine: n = 16 mice; miR-137^flox/+;Nestin-Cre+Vehicle: n = 16 mice; miR-137^flox/+;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-137^flox/+;Nestin-Cre mice demonstrating by the improved number of marbles buried to the same level in miR-137^flox/+ mice (F_3,62 = 14.01, P < 0.0001). Data represent means ± s.e.m; miR-137^flox/++Vehicle: n = 17 mice; miR-137^flox/++Papaverine: n = 16 mice; miR-137^flox/+;Nestin-Cre+Vehicle: n = 17 mice; miR-137^flox/+;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-137^flox/+ and miR-137^flox/+;Nestin-Cre mice during the habituation phase. Left vs. right: miR-137^flox/++Vehicle mice (n = 9 mice), t = 0.2517, P > 0.9999; miR-137^flox/++Papaverine mice (n = 9 mice), t = 2.417, P > 0.9999; miR-137^flox/+;Nestin-Cre+Vehicle mice (n = 10 mice), t = 1.397, P > 0.9999; miR-137^flox/+;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-137^flox/+;Nestin-Cre mice, as indicated by the significantly increased interacting time with a mouse versus empty cage (miR-137^flox/+;Nestin-Cre+Papaverine mice: t_{Empty cage vs. Stranger1} = 3.705, P = 0.0119) or with novel mice (Stranger 2) versus familiar mice (Stranger 1) (miR-137^flox/+;Nestin-Cre+Papaverine mice: t_{Stranger2 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-137^flox/+;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-137^flox/+ or miR-137^flox/+;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-137^flox/+;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-137^flox/+;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-137^flox/+ and miR-137^flox/+;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-137^flox/+ and miR-137^flox/+;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 β-Actin), which associated with the reduced phosphorylation of PKA (P-PKA / PKA-Cα) without changing total PKA levels. After injecting papaverine, the reduced phosphorylation of PKA in miR-137^flox/+;Nestin-Cre mice has resorted to the same level in miR-137^flox/+ 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: F_3,8 = 11.66, P = 0.0027; P-PKA: F_3,8 = 6.085, P = 0.0184; PKA-Cα: F_3,8 = 0.2374, P = 0.8679; P-PKA/PKA-Cα: F_3,8 = 24.66, P = 0.0002. n.s., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 8:. 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-137^flox/+ and miR-137^flox/+;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-137^flox/+;Nestin-Cre mice (Day5: miR-137^flox/+ + sh-Pde10a vs. miR-137^flox/+;Nestin-Cre + sh-Pde10a: t = 2.738, P = 0.2172; miR-137^flox/+ + sh-Neg vs. miR-137^flox/+;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 (F_3,33 = 0.2683, P = 0.8478), it significantly ameliorated the impaired latency to the platform (F_3,33 = 5.377, P = 0.0040) and increased the number of target crossings (F_3,33 = 3.073, P = 0.0411) in miR-137^flox/+;Nestin-Cre mice. Data represent means ± s.e.m; miR-137^flox/++sh-Neg: n = 9 mice; miR-137^flox/++sh-Pde10a: n = 10 mice; miR-137^flox/+;Nestin-Cre+sh-Neg: n = 9 mice; miR-137^flox/+;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-137^flox/+ and miR-137^flox/+;Nestin-Cre mice during the habituation phase. Left vs. right: miR-137^flox/++sh-Neg mice (n = 9 mice), t = 1.446, P > 0.9999; miR-137^flox/++sh-Pde10a mice (n = 10 mice), t = 2.464, P > 0.9999; miR-137^flox/+;Nestin-Cre+sh-Neg mice (n = 9 mice), t = 2.079, P > 0.9999; miR-137^flox/+;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-137^flox/+;Nestin-Cre mice, as indicated by the significantly increased interacting time with a mouse versus empty cage (miR-137^flox/+;Nestin-Cre+sh-Pde10a mice: t_{Empty cage vs. Stranger1} = 6.742, P < 0.0001) or with novel mice (Stranger 2) versus familiar mice (Stranger 1) (miR-137^flox/+;Nestin-Cre+sh-Pde10a mice: t_{Stranger2 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-137^flox/+ and miR-137^flox/+;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-137^flox/+;Nestin-Cre neurons compared with controls, as determined by Sholl analysis (Data represent means ± s.e.m; Interaction: F_57,1340 = 3.286, P < 0.0001; Distance: F_19,1340 = 795.4, P < 0.0001; Treatment: F_3,1340 = 177.5, P < 0.0001. Two-way ANOVA with Bonferroni post hoc test.). sh-Pde10a significantly reduced the dendritic length (miR-137^flox/+;Nestin-Cre mice: t_{sh-Neg vs. sh-Pde10a} = 2.822, P = 0.0354) (g) and the number of dendritic nodes (miR-137^flox/+;Nestin-Cre mice: t_{sh-Neg vs. sh-Pde10a} = 3.485, P = 0.0046) (h) in miR-137^flox/+; 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.