Research ArticleNEURODEVELOPMENT

Disruption of SynGAP–dopamine D1 receptor complexes alters actin and microtubule dynamics and impairs GABAergic interneuron migration

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Science Signaling  06 Aug 2019:
Vol. 12, Issue 593, eaau9122
DOI: 10.1126/scisignal.aau9122
  • Fig. 1 Regulation of D1R-SynGAP complex formation.

    (A and B) Reciprocal coimmunoprecipitation (CO-IP) analysis of D1R with a SynGAP antibody (A) and SynGAP with a D1R antibody (B) in brain lysates from prenatal mice. Blots represent three independent experiments. IB, immunoblot. (C and D) Representative blots (C) and densitometric analysis (D) of the coimmunoprecipitation of D1R and SynGAP in lysates from HEK293T cells transfected with GFP-tagged D1R and SynGAP and treated with the D1R agonist SKF81297 (10 μM for 30 min) alone or with the D1R antagonist SCH23390 (1 μM for 30 min before treatment with SKF81297). Data are means ± SEM, presented as the percentage of the control sample, from n = 3 independent experiments. **P < 0.01 as compared to controls and #P < 0.05 as compared to SKF81297, by one-way ANOVA followed by Tukey’s test. (E and F) Western blot of pulldown of SynGAP from mouse brain lysates incubated overnight ex vivo with (E) GST-D1RIL3 or GST-D1RCT, or (F) GST-D1RIL3-1, GST-D1RIL3-2, GST-D1RIL3-3, or GST-D1RIL3-4. Blots represent three independent experiments performed. (G and H) Representative blots (G) and densitometric analysis (H) of the coimmunoprecipitation of SynGAP with D1R from mouse brain lysates incubated ex vivo with TAT, TAT-D1Rpep, or the D1R agonist SKF81297 (each 10 μM for 30 min). Data are means ± SEM, presented as the percentage of the control and TAT sample, from n = 3 independent experiments. ***P < 0.001 as compared to the TAT and SKF81297 group by two-way ANOVA followed by Bonferroni post hoc test.

  • Fig. 2 Disrupted GABAergic interneuron tangential migration in TAT-D1Rpep–injected embryonic mouse brains.

    (A and B) Coronal sections of E18 or P14 brains immunostained with CB [calbindin-28 (GABAergic interneuron marker)] antibody were straightened and divided into seven equidistant bins representing the tangential migratory pathway of interneurons (A). Fluorescently labeled cell numbers were counted in each bin and normalized to the total CB-positive cell number in all bins (B). Scale bars, 200 μm. Data are means ± SEM of n = 7 (saline), 9 (TAT), or 6 (TAT-D1Rpep) embryonic brains from three pregnant mice. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to saline group in the same bin by two-way ANOVA. (C and D) Cell migration in Matrigel explants from E18 embryos injected with TAT or TAT-D1Rpep from E12 to E17. The migrating distance out of the tissue was measured and normalized to the radius of the tissue. Data are means ± SEM of n = 12 explants from three embryonic brains in each group. ***P < 0.001 as compared to TAT group by t test. (E and F) Cortical explants from E14 embryos were included in Matrigel and treated with saline, TAT, or TAT-D1Rpep, and cell migration was monitored (E). The migrating distance out of the tissue was measured and normalized to the radius of the tissue. Scale bar, 200 μm. Data are means ± SEM of n = 12 to 15 explants from three embryonic brains in each group. Statistical significance was assessed by one-way ANOVA, followed by Dunnett’s test.

  • Fig. 3 Abnormal distribution of both PV and CB interneurons in adult offspring from TAT-D1Rpep–injected pregnant mice.

    (A) Schematic showing the timeline of peptide injection and immunostaining. (B and C) Five sampling ROIs immunolabeled with either PV or CB antibodies were outlined across the neocortex along the medial-lateral axis. The number of PV- and CB-positive cells was counted and presented as a percentage of total cells in the five ROIs. Scale bar, 200 μm. (D) Distribution of PV- and CB-labeled cells in the whole five bins across the neocortex along the medial-lateral axis. Scale bar, 500 μm. (E and F) Statistical significance was assessed for the distribution of PV-labeled (E) and CB-labeled (F) cells in the neocortex along the medial-lateral axis. Data are means ± SEM of n = 8 ROIs from three different mice per group. *P < 0.05 and **P < 0.01 as compared to TAT group by two-way ANOVA, followed by Bonferroni post hoc test. ROI, region of interest; PV, parvalbumin.

  • Fig. 4 SynGAP modulates D1R-mediated signaling and enhances D1R cell surface membrane expression.

    (A) cAMP analysis in HEK293T cells expressing D1R with SynGAP or D1R alone in the presence or absence of SKF81297 (10 μM for 15 min). Results for each sample are presented as the percentage of the control + D1R + pcDNA3 sample. Data are means ± SEM of n = 3 independent experiments. ***P < 0.001 as compared to D1R + pcDNA3 + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (B and C) Representative blots (B) and densitometric analysis (C) of PKA phosphorylation (Thr197) in HEK293T cells expressing D1R with SynGAP and HEK293T cells expressing D1R alone. PKA was used as a loading control. The amount of p-PKA was normalized to that of PKA. Results for each sample are presented as the percentage of the control + D1R + pcDNA3 sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 by two-way ANOVA, followed by Bonferroni post hoc test. (D and E) Representative blots (D) and densitometric analysis (E) of p38 phosphorylation in HEK293T cells expressing D1R with SynGAP and HEK293T cells expressing D1R alone. The amount of p-p38 was normalized to that of p38. Results for each sample are presented as the percentage of the control + D1R + pcDNA3 sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to D1R + pcDNA3 + control group and ***P < 0.001 as compared to D1R + pcDNA3 + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (F) cAMP analysis in primary cultured mouse neurons pretreated with TAT or TAT-D1Rpep in the presence or absence of SKF81297 (10 μM for 15 min). Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. ***P < 0.001 as compared to TAT + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (G and H) Representative blots (G) and densitometric analysis (H) of PKA phosphorylation (T197) in primary cultured mouse neurons treated with TAT or TAT-D1Rpep. The amount of p-PKA was normalized to that of PKA. Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to TAT SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (I and J) Representative blots (I) and densitometric analysis (J) of p38 phosphorylation in primary cultured mouse neurons treated with TAT or TAT-D1Rpep. The amount of p-p38 was normalized to that of p38. Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to TAT SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (K and L) Representative blots (K) and densitometric analysis (L) of cell surface membrane localization of GFP-D1R proteins in HEK293T cells expressing GFP-D1R with SynGAP and HEK293T cells expressing GFP-D1R alone. The amount of biotinylated GFP-D1R was normalized to that of total GFP-D1R. Results for each sample are presented as the percentage of the D1R + pcDNA3 sample. **P < 0.01 as compared to D1R + pcDNA3 group by t test. Data are means ± SEM of n = 3 independent experiments. (M and N) Representative blots (M) and densitometric analysis (N) of cell surface membrane localization of D1R in primary cultured mouse neurons treated with TAT or TAT-D1Rpep. The amount of biotinylated D1R was normalized to that of total D1R. Results for each sample are presented as the percentage of the TAT sample. Data are means ± SEM of n = 3 independent experiments. *P < 0.05 as compared to TAT group by t test.

  • Fig. 5 Disruption of the D1R-SynGAP complex decreases actin at the leading process tips by increasing cortactin proteolysis and decreasing PKA phosphorylation.

    (A to C) TAT- and TAT-D1Rpep–treated interneurons labeled for F-actin in green (phalloidin), cortactin in red (anticortactin), and nuclei in blue (DAPI) (A). The percentage of F-actin–positive tips in all the tips within an entire cell was significantly reduced in TAT-D1Rpep–treated interneurons (B), as well as the number of F-actin–positive tips per cell (C). Data are means ± SEM of n = 15 cells from three independent primary cultures. *P < 0.05 and ***P < 0.001 as compared to TAT group by t test. (D to F) Interneurons transfected with control, SynGAP siRNA, or SynGAP siRNA + siRNA-resistant (siRNA-R) SynGAP were labeled for F-actin in green (phalloidin), cortactin in red (anticortactin), and nuclei in blue (DAPI) (D). The percentage of F-actin–positive tips in all the tips within an entire cell was significantly reduced in SynGAP siRNA–transfected interneurons (E), as well as the number of F-actin–positive tips per cell (F). Data are means ± SEM of n = 15 cells from three independent primary cultures. *P < 0.05 and ***P < 0.001 as compared to control group by one-way ANOVA, followed by Dunnett’s test. (G and H) Representative blots (G) and densitometric analysis (H) of proteolytic cortactin in mouse embryonic brain tissue from TAT- or TAT-D1Rpep–injected groups. The amount of proteolytic cortactin was normalized to that of the loading control α-tubulin. Results for each sample are presented as the percentage of the mean of TAT-injected group. Data are means ± SEM of n = 5 (TAT) or 7 (TAT-D1Rpep) embryonic brains from three pregnant mice. *P < 0.05 compared to TAT group by t test. (I and J) Representative blots (I) and densitometric analysis (J) of PKA phosphorylation (Thr197) in embryonic brain tissue from TAT- or TAT-D1Rpep–injected groups. The amount of phosphorylated PKA was normalized to that of PKA. Results for each sample are presented as the percentage of the mean of TAT-injected group. Data are means ± SEM of n = 5 (TAT) or 7 (TAT-D1Rpep) embryonic brains isolated from three pregnant mice. **P < 0.01 compared to TAT group by t test. (K and L) Representative blots (K) and densitometric analysis (L) of proteolytic cortactin in primary cultured mouse neurons transfected with control siRNA, SynGAP siRNA, or SynGAP siRNA + siRNA-R SynGAP. The amount of proteolytic cortactin was normalized to that of the loading control α-tubulin. Results for each sample are presented as the ratio of SKF81297 to control. Data are means ± SEM of n = 6 independent experiments. **P < 0.01 compared to control siRNA group by one-way ANOVA, followed by Dunnett’s test. (M and N) Representative blots (M) and densitometric analysis (N) of PKA phosphorylation (Thr197) in primary cultured mouse neurons transfected with control siRNA, SynGAP siRNA, or SynGAP siRNA + siRNA-R SynGAP. PKA was used as a loading control. The amount of phosphorylated PKA was normalized to that of PKA. Results for each sample are presented as the ratio of SKF81297 to control. Data are means ± SEM of n = 6 independent experiments. ***P < 0.001 as compared to control siRNA group by one-way ANOVA, followed by Dunnett’s test.

  • Fig. 6 Disruption of the D1R-SynGAP complex changes neuronal cytoskeleton by decreasing MAP2 phosphorylation.

    (A to C) Primary cultured MGE-derived cells were treated with TAT or TAT-D1Rpep, and immunostaining was performed to analyze distribution of the motor protein cytoplasmic dynein heavy chain (CDHC; green) or the cytoskeletal protein neuronal β III-tubulin (TuJ1; red). Nuclei were labeled with the DNA stain DAPI (blue) (A). Quantification of the immunolabeling intensity from soma to leading process tip for CDHC (B) and TuJ1 (C). Images represent 15 neurons from three independent experiments performed. (D to F) Primary cultured MGE-derived cells were transfected with control, SynGAP siRNA, or SynGAP siRNA + siRNA-R SynGAP, and immunostaining was performed to analyze distribution of the motor protein CDHC (green) or the cytoskeletal protein neuronal TuJ1 (red). Nuclei were labeled with the DNA stain DAPI (blue) (D). Quantification of the immunolabeling intensity from soma to leading process tip for CDHC (E) and TuJ1 (F). Images represent 15 neurons from three independent experiments performed. (G and H) Representative blots (G) and densitometric analysis (H) of MAP2 phosphorylation in mouse brain slices treated with TAT or TAT-D1Rpep (10 μM for 30 min before SKF81297 treatment) in the absence or presence of SKF81297 (10 μM for 30 min). The amount of p-MAP2 was normalized to that of MAP2. Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. **P < 0.01 as compared to TAT group and ***P < 0.001 as compared to TAT + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test. (I to K) Representative blots (I) and densitometric analysis (J and K) of ERK1/2 phosphorylation in mouse brain slices treated with TAT or TAT-D1Rpep (10 μM for 30 min before SKF81297 treatment) in the absence/presence of SKF81297 (10 μM for 30 min). The amount of p-ERK1(/2) was normalized to that of ERK1(/2). Results for each sample are presented as the percentage of the control + TAT sample. Data are means ± SEM of n = 3 independent experiments. ***P < 0.001 as compared to TAT + SKF81297–treated group by two-way ANOVA, followed by Bonferroni post hoc test.

  • Fig. 7 Schematic diagram of interaction between D1R and SynGAP.

    SynGAP enhances D1R membrane expression and D1R-mediated signaling pathway, including cAMP accumulation, phosphorylation of PKA, p38, and ERK1/2, through the D1R-SynGAP interaction. Increased phospho-PKA leads to decreased cortactin proteolysis and thus increases actin polymerization. Increased phosphorylated ERK1/2 results in increased MAP2 and thus increases cytoplasmic dynein motor protein-tubulin interaction. Both changes in actin and microtubule dynamics lead to enhanced GABAergic interneuron tangential migration.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/593/eaau9122/DC1

    Fig. S1. Schematic representation of generated GST fusion proteins encoding truncated D1RIL3 segments.

    Fig. S2. TAT fusion peptides are able to cross both the placenta and blood-brain barrier, and TAT-D1Rpep is able to disrupt the D1R-SynGAP interaction.

    Fig. S3. Cell migration is delayed in cultured MGE explants treated with TAT-D1Rpep in vitro.

    Fig. S4. TAT-D1Rpep disrupts the D1R-SynGAP interaction in cultured MGE neurons.

    Fig. S5. TAT-D1Rpep disrupts the D1R-SynGAP interaction in cultured glutamatergic neurons.

    Fig. S6. SynGAP knockdown decreases the effects of D1R activation on cortactin proteolysis and PKA phosphorylation.

  • This PDF file includes:

    • Fig. S1. Schematic representation of generated GST fusion proteins encoding truncated D1RIL3 segments.
    • Fig. S2. TAT fusion peptides are able to cross both the placenta and blood-brain barrier, and TAT-D1Rpep is able to disrupt the D1R-SynGAP interaction.
    • Fig. S3. Cell migration is delayed in cultured MGE explants treated with TAT-D1Rpep in vitro.
    • Fig. S4. TAT-D1Rpep disrupts the D1R-SynGAP interaction in cultured MGE neurons.
    • Fig. S5. TAT-D1Rpep disrupts the D1R-SynGAP interaction in cultured glutamatergic neurons.
    • Fig. S6. SynGAP knockdown decreases the effects of D1R activation on cortactin proteolysis and PKA phosphorylation.

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