Interplay between DISC1 and GABA signaling regulates neurogenesis in mice and risk for schizophrenia.

DISC1 KD-induced precocious dendritic growth of newborn neurons in the adult dentate gyrus requires GABA-induced depolarization(A) A schematic diagram of the experimental design and sample projected confocal images of newborn neurons with retrovirus-mediated co-expression of GFP and a control shRNA (C1), shRNA-DISC1 (D1), or shRNA-NKCC1 (NK1) in the adult mouse dentate gyrus at 14 dpi. Scale bar: 50 µm.(B) Summary of total dendritic length of GFP+ neurons under different conditions. Values represent mean ± SEM (n = 4 animals with a total of 29–36 neurons for each condition; *: P < 0.05; ANOVA).(C–F) Effect of retrovirus-mediated double NKCC1 and DISC1 KD on dendritic development and AKT/mTOR signaling in newborn neurons in the adult dentate gyrus. Shown in (C) are schematic diagrams of retroviral vectors and experimental design, and sample projected confocal images of a newborn neuron with double KD at 14 dpi. Shown are summaries of total dendritic length (D) and quantifications of pAKT (E) and pS6 (F) levels in the cytosol of new neurons at 14 dpi. Numbers associated with the bar graph represent total numbers of neurons examined under each condition. Values represent mean ± SEM (n = 4 animals for each condition; *: P < 0.05; ANOVA).

DISC1 KD-induced precocious dendritic development of newborn neurons in the adult dentate gyrus is enhanced by activation of GABA signaling(A–D) Effect of vigabatrin (VGA) on dendritic development and AKT/mTOR signaling in newborn neurons in the adult hippocampus with or without DISC1 KD. Shown in (A) are the schematic diagram of the experimental design and sample projected confocal images of new neurons with retrovirus-mediated co-expression of GFP and a control shRNA (C1) or shRNA-DISC1 (D1), with or without VGA injection (25 µg/g body weight per day from 3 to 6 dpi, i.p.) and examined at 7 dpi. Scale bar: 50 µm. Shown are summaries of total dendritic length (B) and quantifications of pAKT (C) and pS6 (D) levels in the cytosol of newborn neurons at 7 dpi. Values represent mean ± SEM (n = 4 animals; *: P < 0.05; n.s.: P > 0.1; ANOVA).(E–H) Same as in (A–D), except that pentobarbital (25 µg/g per day from 6 to 10 dpi; i.p.) was injected and analysis was performed at 11 dpi.(H–K) Same as in (B–D), except that newborn neurons expressing shRNA-control (C1), KIAA1212 (K1), shRNA-NK1 (NK1), or both K1 and NK1 were analyzed at 14 dpi.(L) A model of interaction between depolarizing GABA and DISC1 signaling in regulating dendritic growth of newborn neurons in the adult dentate gyrus.

DISC1 KD fails to affect development of newborn dentate granule neurons during early postnatal hippocampal neurogenesis(A) A schematic diagram of the experimental design and sample projected confocal images of newborn neurons with retrovirus-mediated co-expression of GFP and a control shRNA (C1), or shRNA-DISC1 (D1), in the dentate gyrus of neonatal mouse hippocampus. Scale bar: 50 µm.(B–D) Summary of the mean total dendritic length (B), soma size (C), primary dendrite number (D) of GFP+ neurons under different conditions. The same data on the time course of dendritic development of shRNA-C1/GFP+ neurons in the adult hippocampus (P42) as in Figure 1B are replotted in (B) for direct comparison. Values represent mean ± SEM (n = 4–6 animals with a total of 36–45 neurons for each condition; P > 0.05; ANOVA).(E) Positioning of newborn neurons in the dentate gyrus under different conditions. Shown is the summary of the distribution of soma of GFP+ neurons within the four domains in the dentate gyrus (as defined in Figure S1C). The same group of neurons as in (B–D) was used.

Newborn neurons during adult and early postnatal neurogenesis exhibit differential tempo in shifting the polarity of GABA responses(A) A schematic diagram of the experimental design and sample Ca2+ imaging analysis of new neurons expressing RFP and shRNA-C1 in P42 animals. Sample confocal images of RFP and Ca2+ dye Fluo4-AM, as well as Ca2+ responses were shown at the basal level, followed by application of GABAAR agonist muscimol (musc.; 10 µM), and then Ca2+ ionophore ionomycin (iono.; 10 µM). White arrowheads point to RFP+Fluo4+ newborn cells. Scale bar: 20 µm.(B) Summary of mean peak Ca2+ responses to muscimol for newborn neurons expressing shRNA-C1 (C1), shRNA-NKCC1 (NK1), or shRNA-D1 (D1) in the adult dentate gyrus. The values of Ca2+ responses are normalized to the mean fluorescence intensity measured at the baseline condition (set as 0%) and after the ionomycin treatment (set as 100%) in the same cell. Values represent mean ± SEM (n = 6–7 cells; *: P < 0.01; ANOVA).(C and D) Same as (A and B), except that retroviruses co-expressing RFP and shRNA-C1 (C1), shRNA-KCC2 (K2), or shRNA-D1 (D1) were injected into P10 animals.

DISC1 KD induces precocious dendritic development during early postnatal neurogenesis in the presence of extended period of GABA-induced depolarization(A) A schematic diagram of the experimental design.(B) Sample confocal images of newborn neurons after stereotaxic injection of a mixture of retroviruses co-expressing shRNA-DISC1 (D1)/DsRed and those co-expressing shRNA-KCC2 (K2)/GFP into the dentate gyrus of the P10 animal and analyzed at 7 dpi. Scale bar: 20 µm.(C–D) Summary of the mean total dendritic length of newborn neurons at 7 dpi under different conditions. Rapamycin (20 mg/kg body weight) or saline was i.p. injected daily after viral injection in (D). Values represent mean ± SEM (n = 6–8 animals with a total of 31–50 neurons for each condition; *: P < 0.05; ANOVA).(E– F) Summary models of interaction between DISC1 and depolarizing GABA in regulating dendritic development in a context-dependent fashion. Shown in (E) is a schematic diagram of interaction between depolarizing GABA and DISC1 signaling in regulating dendritic development. Shown in (F) is a model of DISC1 signaling in regulating neuronal development during early postnatal and adult hippocampal neurogenesis and its temporal constraints.

Maternal deprivation stress synergizes with DISC1 KD in regulating early postnatal hippocampal neurogenesis(A) A diagram of the experimental design.(B) Sample confocal images of new neurons at 7 dpi after retrovirus-mediated expression of control shRNA (C1), shRNA-DISC1 (D1), shRNA-NKCC1 (NK1), or co-expression of D1 and NK1, in P10 dentate gyrus with or without maternal deprivation stress. Scale bar: 50 µm.(C–F) Summaries of mean total dendritic length (C and E) and Sholl analysis (D and F) of new neurons at 7 dpi in the early postnatal dentate gyrus. Rapamycin (20 mg/kg body weight) was i.p. injected daily after viral injection for some animals. Values represent mean ± SEM (n = 4–8 animals; P < 0.01; ANOVA).

DISC1 and NKCC1 interact epistatically to affect risk for schizophrenia(A) Haploview LD plot (R-Squared; based on the Scottish sample) of SNPs on the Illumina chip in DISC1 showing interactions with SNPs in NKCC1.(B) Linkage disequilibrium (R-Squared measure) of SNPs in NKCC1 (HapMAP CEU data) showing two SNPs on the Illumina chip that were used for the interaction analysis with DISC1.(C) Interaction between rs10089 (NKCC1) and rs1000731 (DISC1) and risk for schizophrenia in three independent case-control samples of European ancestry.