Absence of BRINP1 in mice causes increase of hippocampal neurogenesis and behavioral alterations relevant to human psychiatric disorders

Abstract

Background: We have previously identified BRINP (BMP/RA-inducible neural-specific protein-1, 2, 3) family genes that possess the ability to suppress cell cycle progression in neural stem cells. Of the three family members, BRINP1 is the most highly expressed in various brain regions, including the hippocampus, in adult mice and its expression in dentate gyrus (DG) is markedly induced by neural activity. In the present study, we generated BRINP1-deficient (KO) mice to clarify the physiological functions of BRINP1 in the nervous system.

Results: Neurogenesis in the subgranular zone of dentate gyrus was increased in BRINP1-KO mice creating a more immature neuronal population in granule cell layer. The number of parvalbumin expressing interneuron in hippocampal CA1 subregion was also increased in BRINP1-KO mice. Furthermore, BRINP1-KO mice showed abnormal behaviors with increase in locomotor activity, reduced anxiety-like behavior, poor social interaction, and slight impairment of working memory, all of which resemble symptoms of human psychiatric disorders such as schizophrenia and attention-deficit/hyperactivity disorder (ADHD).

Conclusions: Absence of BRINP1 causes deregulation of neurogenesis and impairments of neuronal differentiation in adult hippocampal circuitry. Abnormal behaviors comparable to those of human psychiatric disorders such as hyperactivity and poor social behavior were observed in BRINP1-KO mice. These abnormal behaviors could be caused by alteration of hippocampal circuitry as a consequence of the lack of BRINP1.

Figure 1

Targeted disruption of BRINP1 gene in ES cell and mouse. (A) Construct of the targeting vector for homologous recombination to yield BRINP1-KO mice. The coding region of Brinp1 exon8 (filled box) was disrupted by PGK-neomycin resistance cassette. The probe used for Southern blot analysis is shown together with predicted sizes of hybridizing fragments. Sites of restriction enzymes: Av, AvrII; B, BamHI; H, HindIII; Ps, PstI; RV, EcoRV; Xc, XcmI; Xm, XmaI. (B) Southern blot analysis of BamHI-digested genomic DNA extracted from control (TT2) and positive clone (1–9) of ES cells, and F1 mice produced by crossing chimera mice with C57BL/6J mice. (C) Southern blot analysis of genomic DNA extracted from wild-type (+/+) and BRINP1-KO (−/−) mice. (D) mRNA expression of BRINP family genes in BRINP1-KO mice. Total RNA extracted from adult brain of wild-type (+/+), BRINP1 heterozygous (+/−), and BRINP1-KO (−/−) mice were hybridized with each of Brinp1, Brinp2 and Brinp3 antisense probes. Uniform transfer of RNA was confirmed by methylene blue staining.

Figure 2

General health and neurological screening of BRINP1-KO mice. (A) Body weight of BRINP1-KO mice was reduced to about 85% of wild-type mice. (B) Body temperature was measured at rectum. (C) Grip strength of forelimb in Newton. (D) Latency to fall (s) showed in wire hang test. (E) Hot plate test at 55°C. (F) Rotarod test. (wild-type mice, n = 11; BRINP1-KO mice, n = 9) Error bars indicate SEM.

Figure 3

Exploratory activity in BRINP1-KO mice in open field test. (A) Total distance traveled. (B) Counts of vertical activity. (C) Time spent in the center of the compartment. (D) Counts of stereotypic behavior. (wild-type mice, n = 11; BRINP1-KO mice, n = 9) Error bars indicate SEM.

Figure 4

Anxiety-related behavior of BRINP1-KO mice in light/dark transition and elevated plus maze. (A-D) Light/dark transition, (E-H) Elevated plus maze test. (A) Total distance traveled in the light and dark boxes. (B) Time the mice stayed in the light box. (C) Number of transitions between light and dark boxes. (D) Latency time before the first entry into the light box. (E) Number of entries into each open and closed arm. (F) Percent of entries into the open arms. (G) Total distance traveled. (H) Percent time spent on the open arms. (wild-type mice, n = 11; BRINP1-KO mice, n = 9) Error bars indicate SEM.

Figure 5

Social interaction of BRINP1-KO mice in novel environment. (A) Total duration of contacts. (B) Total number of contacts. (C) Total duration of active contacts which either of the mice traveled longer than 5 cm. (D) Mean duration per contacts. (E) Total distance traveled. (wild-type mice, n = 5; BRINP1-KO mice, n = 4) Error bars indicate SEM.

Figure 6

Working memory of BRINP1-KO mice in T-maze test. (A) Percentage of correct responses in free-choice run. (B) Latency to complete one session. (C) Distance traveled within one session. (D) Percentage of correct responses when each delay duration was inserted between forced-choice run and free-choice run. (wild-type mice, n = 11; BRINP1-KO mice, n = 9) Error bars indicate SEM.

Figure 7

Adult neurogenesis is increased in BRINP1-KO mice. Representative images of BrdU positive cells (green) (A,B) and Ki-67 positive cells (red) (C,D) in dentate gyrus of wild-type mice (A,C) and BRINP1-KO mice (B,D) at 8 weeks. DAPI in blue. (E) Representative graph of the number of BrdU-positive and Ki-67-positive cells in each 7 and 8-week-old mouse dentate gyrus. Data are shown as mean ± SEM. (n = 35 sections per genotype; **: p < 0.01, ***: p < 0.001 Wilcoxon rank-sum test). Scale bar; 100 μm.

Figure 8

Neuronal differentiation marker expression in dentate gyrus. (A,B) Doublecortin, (C,D) Calretinin, (E,F) Calbindin expression in 7-week-old mouse dentate gyrus. (A,C,E) Wild-type mice, (B,D,F) BRINP1-KO mice. Scale bar; 50 μm.

Figure 9

Parvalbumin expressing interneurons in BRINP1-KO mice. Representative images of parvalbumin immunoreactivity (green) in hippocampus of wild-type mice (A-C) and BRINP1-KO mice (D-F). DAPI in blue. (G) The number of parvalbumin expressing cells/section is increased in CA1 subregion of BRINP1-KO mice in hippocampus. Data are shown as mean ± SEM. (n = 26-28 sections from 3 different mice per genotype; ***: p < 0.001 Wilcoxon rank-sum test). Scale bar; 100 μm.