Prevention of Neurite Spine Loss Induced by Dopamine D2 Receptor Overactivation in Striatal Neurons

Abstract

Psychosis has been considered a disorder of impaired neuronal connectivity. Evidence for excessive formation of dopamine D2 receptor (D2R) - disrupted in schizophrenia 1 (DISC1) complexes has led to a new perspective on molecular mechanisms involved in psychotic symptoms. Here, we investigated how excessive D2R-DISC1 complex formation induced by D2R agonist quinpirole affects neurite growth and dendritic spines in striatal neurons. Fluorescence resonance energy transfer (FRET), stochastic optical reconstruction microscopy (STORM), and cell penetrating-peptide delivery were used to study the cultured striatal neurons from mouse pups. Using these striatal neurons, our study showed that: (1) D2R interacted with DISC1 in dendritic spines, neurites and soma of cultured striatal neurons; (2) D2R and DISC1 complex accumulated in clusters in dendritic spines of striatal neurons and the number of the complex were reduced after application of TAT-D2pep; (3) uncoupling D2R-DISC1 complexes by TAT-D2pep protected neuronal morphology and dendritic spines; and (4) TAT-D2pep prevented neurite and dendritic spine loss, which was associated with restoration of expression levels of synaptophysin and PSD-95. In addition, we found that Neuropeptide Y (NPY) and GSK3β were involved in the protective effects of TAT-D2pep on the neurite spines of striatal spiny projection neurons. Thus, our results may offer a new strategy for precisely treating neurite spine deficits associated with schizophrenia.

Keywords: D2R–DISC1 complex; GABA; Neuropeptide Y; cell penetrating-peptide; schizophrenia; synaptic spine.

FIGURE 1

Interaction of D2R–DISC1 in single dendritic spines by STORM analysis. (A) Two-color STORM combined with bright field images show the interaction between D2R (green) and DISC1 (red) in single spines. Upper panels show representative images of D2R–DISC1 complex formation in single dendritic spines. Scale bar = 1 μm. Lower panels show magnified representative images of single D2R–DISC1 complexes. Scale bar = 200 nm. (B) Diameter of D2R and DISC1 nanoclusters. (C) Quantification of the distance between D2R and DISC1 nanoclusters in dendritic spines by the nearest neighbor algorithm. (D,E) Number of D2R and DISC1 nanoclusters in single dendritic spines. **P < 0.01 versus control; ****P < 0.0001 versus control; ^##P < 0.01 versus quinpirole; ^####P < 0.001 versus quinpirole by one-way analysis of variance (ANOVA) with post hoc Tukey test.

FIGURE 2

TAT-D2pep protects synaptic spine density and dendritic complexity against D2R hyperactivity in cultured striatal neurons. (A) Representative images of striatal neurons immunostained with MAP-2. Scale bar = 50 μm. (B) Effect of TAT-D2pep on neurite length. TAT-D2pep inhibited the decrease in neurite length of striatal neurons. (C,D) Sholl analysis of cultured striatal neurons treated with quinpirole and TAT-D2pep. Representative images of striatal neurons with superimposed circles at given distances from the center of the soma. For Sholl analysis, the total number of neurite crossings was counted at each circle with the radius increasing in steps of 5 μm. **P < 0.01 versus control; ^##P < 0.01 versus quinpirole. (E) Representative images with high magnification of dendrites immunostained with phalloidin-actin Scale bar = 50 μm (top panels) and 10 μm (bottom panels). (F) quantification of spine density. **P < 0.01 and ***P < 0.001 versus control, ^#P < 0.05 and ^###P < 0.001 versus quinpirole by one-way analysis of variance (ANOVA) with post hoc Tukey test.

FIGURE 3

TAT-D2pep specifically blocks excessive D2R–DISC1 complex formation induced by D2R hyperactivity. (A–C) FRET analysis showed that TAT-D2pep decreased D2R–DISC1 complex formation induced by quinpirole in soma and neurites of striatal neurons. In (A), the boxes in the FRET images are enlarged in the panels to the right. (D,E) FRET images of HEK-293 cells transfected with D2R-EGFP and DISC1-mCherry. D2R hyperactivity-induced D2R–DISC1 complex formation was abolished by TAT-D2pep but not control peptide. Scale bar = 10 μm. (F) Schematic relationship between FRET and nanocluster distance: r₁: radius of D2R nanocluster; r₂: radius of DISC1 nanocluster. If the D2R–DISC1 nanocluster distance is below the sum of r₁ + r₂ + 10 nm, FRET will occur; If the D2R–DISC1 nanocluster distance is above the sum of r₁ + r₂ + 10 nm, FRET will not be detected. Data are shown as means ± SEM and normalized; **P < 0.01 and ****P < 0.0001 versus control, ^##P < 0.01, ^###P < 0.001 and ^####P < 0.0001 versus quinpirole by one-way analysis of variance (ANOVA) with post hoc Tukey test; n > 30 cells per group.

FIGURE 4

TAT-D2pep blocks D2R hyperactivity-induced down-regulation of synaptophysin and PSD-95. (A–C) Western blot analysis showed that TAT-D2pep but not TAT-D2pep-NC increased expression of synaptophysin and PSD-95 in HEK-293 cells co-expressing D2R-EGFP and DISC1 mCherry; n > 3 per group. (D,E) Representative images of striatal neurons co-immunostained with MAP-2 and synaptophysin. Pearson’s correlation coefficients values for MAP-2 and synaptophysin colocalization assays. (F,G) Representative images of striatal neurons co-immunostained with MAP-2 and PSD-95. Pearson’s correlation coefficients values for MAP-2 and PSD-95 colocalization assays. Scale bars = 50 μm and 15 μm (enlarged), **P < 0.01 and ***P < 0.001 versus control, ^#P < 0.05 and ^###P < 0.001 versus quinpirole by one-way analysis of variance (ANOVA) with post hoc Tukey test.

FIGURE 5

TAT-D2pep protects neurites through upregulating NPY in striatal GABAergic neurons. (A,B) Western blot analysis showed that TAT-D2pep but not TAT-D2pep-NC inhibited downregulation of NPY in HEK-293 cells expressing D2R-EGFP and DISC1 mCherry; n > 3 per group. (C,D) Triple-immunostaining for phalloidin-actin, NPY and GAD67 in primary striatal neurons. TAT-D2pep blocked quinpirole-induced downregulation of NPY in striatal GABAergic neurons, accompanied by normal neurite growth. *P < 0.05 and ***P < 0.001 versus control, ^#P < 0.05 and ^###P < 0.001 versus quinpirole by one-way analysis of variance (ANOVA) with post hoc Tukey test.

FIGURE 6

TAT-D2pep protects neurites through increasing phosphorylation of GSK3β in striatal GABAergic neurons. (A,B) Western blot analysis showed that TAT-D2pep increased downregulation of pGSK3β in HEK-293 cells expressing D2R-EGFP and DISC1 mCherry; n > 3 per group. (C,D) Triple-immunostaining for phalloidin-actin, pGSK3β and GAD67 in primary striatal neurons. TAT-D2pep blocked quinpirole-induced downregulation of pGSK3β in striatal GABAergic neurons, accompanied by normal neurite growth. *P < 0.05 and ***P < 0.001 versus control, ^#P < 0.05 and ^###P < 0.001 versus quinpirole by one-way analysis of variance (ANOVA) with post hoc Tukey test.