Behavioral characterization of mice overexpressing human dysbindin-1

Abstract

Background: The dysbindin-1 gene (DTNBP1: dystrobrevin binding protein 1) is a promising schizophrenia susceptibility gene, known to localize almost exclusively to neurons in the brain, and participates in the regulation of neurotransmitter release, membrane-surface receptor expression, and synaptic plasticity. Sandy mice, with spontaneous Dtnbp1 deletion, display behavioral abnormalities relevant to symptoms of schizophrenia. However, it remains unknown if dysbindin-1 gain-of-function is beneficial or detrimental.

Results: To answer this question and gain further insight into the pathophysiology and therapeutic potential of dysbindin-1, we developed transgenic mice expressing human DTNBP1 (Dys1A-Tg) and analyzed their behavioral phenotypes. Dys1A-Tg mice were born viable in the expected Mendelian ratios, apparently normal and fertile. Primary screening of behavior and function showed a marginal change in limb grasping in Dys1A-Tg mice. In addition, Dys1A-Tg mice exhibited increased hyperlocomotion after methamphetamine injection. Transcriptomic analysis identified several up- and down-regulated genes, including the immediate-early genes Arc and Egr2, in the prefrontal cortex of Dys1A-Tg mice.

Conclusions: The present findings in Dys1A-Tg mice support the role of dysbindin-1 in psychiatric disorders. The fact that either overexpression (Dys1A-Tg) or underexpression (Sandy) of dysbindin-1 leads to behavioral alterations in mice highlights the functional importance of dysbindin-1 in vivo.

Figure 1

Generation of Dys1A-Tg mice. (A) Schematic of the transgene construct (hDTNBP1-GFP) with CA promoter, human dysbindin-1 (hDTNBP1) cDNA C-terminally fused to GFP, and Simian virus 40 polyadenylation signal sequence (PolyA). (B) Western blot analysis of transgenically expressed human dysbindin-1 and endogenous (mouse) dysbindin-1 protein in Dys1A-Tg mice. Protein lysates prepared from whole brain of adult male Dys1A-Tg mice (lines 1 and 2) and wild-type littermates, a Sandy mouse lacking dysbindin-1 protein, and a wild-type control mouse were subjected to western blot analysis with anti-dysbindin and anti-GAPDH antibodies. Closed and open arrowheads indicate transgene products and endogenous dysbindin-1 protein, respectively. (C) The intensity of each band in the western blot (B) was quantitated and normalized vs. GAPDH. Data are expressed as mean ± SEM. WT, wild-type; Tg, Dys1A-Tg; KO, knockout. *P < 0.05, ***P < 0.001 vs. wild-type of the same line. (D) Semi-quantitative RT-PCR analysis of transgenic human dysbindin-1 and endogenous mouse dysbindin-1 mRNA expression in various tissues of Dys1A-Tg mice and wild-type littermates. GAPDH serves as an internal control. hDTNBP1, human dysbindin-1; mDtnbp1, mouse dysbindin-1; SM, smooth muscle; PFC, prefrontal cortex; HP, hippocampus.

Figure 2

Essentially normal behavior in Dys1A-Tg mice under basal conditions. Distance traveled (A), vertical rearing activity (B), and time spent in the center area (C) of the open-field test, PPI of the acoustic startle response (D), and latency to fall in the accelerated rotarod test (E) were analyzed in Dys1A-Tg (closed symbols and bars) and wild-type (open symbols and bars) mice. Data are expressed as mean ± SEM. Number of mice for each genotype, 19–21 (A–C), 17–19 (D), and 17 (E). Statistical analysis was performed by repeated two-way ANOVA. **P < 0.01 vs. PPI value at 68 db of pre-pulse intensity. There was no significant main effect of genotype in any experiment.

Figure 3

Slightly increased responses to METH and PCP in Dys1A-Tg mice. Acute behavioral responses to METH (A and B) or PCP (C and D) were examined using locomotor (A and C), PPI (B), and object investigation (D) tests in Dys1A-Tg (closed symbols and bars) and wild-type (open symbols and bars) mice. METH or PCP were injected at 60 min and cumulative locomotor activity measured for 70–100 min was indicated in bar graphs in (A) and (C). Data are expressed as mean ± SEM. Number of mice for each genotype, 14–27 (A), 17–35 (B), 3–8 (C), and 3–5 (D). Statistical analysis was performed by repeated three or two-way ANOVA followed by the Tukey–Kramer post-hoc test. *P < 0.05, **P < 0.01 vs. vehicle of the same genotype. ^# P < 0.05 vs. wild-type of the same treatment.

Figure 4

Effects of chronic PCP administration in Dys1A-Tg mice. (A) The experimental schedule consisted of four successive behavioral tests (see Methods for details). (B–E) PCP was administered subcutaneously daily for 14 days, and locomotor activity measured using a digital counter system with an infrared sensor for 90 min (B), immobility time in the FST (C), duration of social interaction between two unfamiliar test mice of the same genotype and treatment (D), and object exploration time and preference index in the test session of the novel object recognition memory test on day 21 (E) were determined in Dys1A-Tg (closed bars) and wild-type (open bars) mice. Data are expressed as mean ± SEM. Number of mice for each genotype is 12–14 obtained from three independent cohorts. Statistical analysis was performed by two-way ANOVA followed by the Tukey–Kramer post hoc test. *P < 0.05 vs. vehicle of the same genotype, ^# P < 0.05 vs. wild-type of the same treatment.