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. 2016 Mar 31:7:22.
doi: 10.1186/s13229-016-0079-7. eCollection 2016.

Brinp1(-/-) mice exhibit autism-like behaviour, altered memory, hyperactivity and increased parvalbumin-positive cortical interneuron density

Susan R Berkowicz  1 , Travis J Featherby  2 , Zhengdong Qu  3 , Aminah Giousoh  1 , Natalie A Borg  1 , Julian I Heng  3 , James C Whisstock #  1   4 , Phillip I Bird #  1
Affiliations

Brinp1(-/-) mice exhibit autism-like behaviour, altered memory, hyperactivity and increased parvalbumin-positive cortical interneuron density

Susan R Berkowicz et al. Mol Autism. .

Abstract

Background: BMP/RA-inducible neural-specific protein 1 (Brinp1) is highly conserved in vertebrates, and continuously expressed in the neocortex, hippocampus, olfactory bulb and cerebellum from mid-embryonic development through to adulthood.

Methods: Brinp1 knock-out (Brinp1(-/-)) mice were generated by Cre-recombinase-mediated removal of the third exon of Brinp1. Knock-out mice were characterised by behavioural phenotyping, immunohistochemistry and expression analysis of the developing and adult brain.

Results: Absence of Brinp1 during development results in a behavioural phenotype resembling autism spectrum disorder (ASD), in which knock-out mice show reduced sociability and changes in vocalisation capacity. In addition, Brinp1(-/-) mice exhibit hyper-locomotor activity, have impaired short-term memory, and exhibit poor reproductive success. Brinp1(-/-) mice show increased density of parvalbumin-expressing interneurons in the adult mouse brain. Brinp1(-/-) mice do not show signs of altered neural precursor proliferation or increased apoptosis during late embryonic brain development. The expression of the related neuronal migration genes Astn1 and Astn2 is increased in the brains of Brinp1(-/-) mice, suggesting that they may ameliorate the effects of Brinp1 loss.

Conclusions: Brinp1 plays an important role in normal brain development and function by influencing neuronal distribution within the cortex. The increased cortical PV-positive interneuron density and altered behaviour of Brinp1(-/-) mice resemble features of a subset of human neurological disorders; namely autism spectrum disorder (ASD) and the hyperactivity aspect of attention deficit hyperactivity disorder (ADHD).

Keywords: Autism spectrum disorder; Brinp1; Cortex; Hyperactivity; Interneuron; Knock-out; Neurodevelopment; Parvalbumin.

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Figures

Fig. 1
Fig. 1
BRINP1 and ASTN2 share homology and a common locus. a Schematic of the BRINP1/ASTN2 locus at 9q33.1. b BRINP1 and ASTN2 show 20 % homology via a common MACPF domain. Sig = Signal sequence, E = EGF-like domain, FNIII = Fibronectin III domain
Fig. 2
Fig. 2
Brinp1 targeting. a The Brinp1 targeting vector was designed with a neomycin resistance cassette after exon 3, and FRT sites positioned before and after the neor cassette. The 190 bp third exon of Brinp1 contains the start of the MACPF domain. LoxP sites flank exon 3 and the neor cassette. When crossed with a mouse line expressing Cre-recombinase, the recombination of LoxP sites resulted in the deletion of exon 3 and the neor cassette. b Genomic DNA isolated from the spleen was cleaved with Pst I and Bgl III and hybridised to 500 bp genomic DNA probes from the 5′ region (Pst I) and 3′ region (Bgl III) of the targeting construct. In wild-type DNA, species of 7.1 kb (Pst I) and 18.4 kb (Bgl III) were detected. These products were not present in DNA from Brinp1 −/− mutants, replaced with shorter species of 5.4 kb (Pst I) and 11.4 kb (Bgl III). c cDNA from the brain tissue of WT or Brinp1 −/− mice was tested for exon 3 deletion by RT-PCR. Primers designed to regions of exon 2 and exon 6 resulted in a PCR product size corresponding to the removal of the 190 bp exon 3 for Brinp1 −/− cDNA. d Sequencing of the Brinp1 −/− allele RT-PCR product showed the expected absence of exon 3, and that splicing fuses exons 2 and 4, resulting in a frame shift that introduces a stop codon after 24 residues. e By immunoblotting, full-length 85 kDa BRINP1 was present in lysates of mouse brains at postnatal day 12 and absent in Brinp1 −/− mice
Fig. 3
Fig. 3
Reduced litter survival and postnatal growth of Brinp1 knock-out mice. a Breeders were monitored for litter size at birth and at age of weaning (P21). i) No significant differences in number of pups per litter at postnatal day 0, from WT × WT, het × het, Brinp1 −/− × Brinp1 −/−, or WT × Brinp1 −/− parents, F(3,62) = 0.1624, p = 0.9212, one-way ANOVA. ii) The knock-out allele present in breeders impacted the number of pups weaned at postnatal day 21, F(3,62) = 9.119, p < 0.001, one-way ANOVA. Tukey HSD multiple comparison tests showed significant differences: WT × WT and het × het: p = 0.122, WT × WT and Brinp1 −/− × Brinp1 −/−: p < 0.001, WT × WT and WT × Brinp1 −/−: p = 0.006, het × het and Brinp1 −/− × Brinp1 −/−: p = 0.018, het × het and WT × Brinp1 −/−: p = 0.588, Brinp1 −/− × Brinp1 −/− and WT × Brinp1 −/−: p = 0.377. b Brinp1 −/− mice weighed from week 3 to week 12. i) Female Brinp1 −/− mice showed a significant reduction in body weight by repeat measures two-way ANOVA: F(2,18) = 27.580, p < 0.001. A Tukey HSD multiple comparison test found female Brinp1 −/− mice to weight significantly less than WT littermates of the same sex (p < 0.001). Brinp1 het female mice were also found to weigh significantly less than WT (p < 0.001). No significant effect of genotype was found between female Brinp1 het and Brinp1 −/− mice (p = 0.362). ii) Male Brinp1 −/− mice also show a significant reduction in body weight by repeat measures two-way ANOVA: F(2,23) = 8.312, p = 0.002. A Tukey HSD multiple comparison test found a significant difference between male WT and Brinp1 −/− mice (p = 0.002) and male Brinp1 het and Brinp1 −/− mice (p = 0.013). No significant effect of genotype found between male WT and Brinp1 het mice (p = 0.704). Results represented as the mean ± SD *p < 0.05, **p < 0.0.1, ***p < 0.001, ****p < 0.0001
Fig. 4
Fig. 4
Altered social interaction and vocalisation of Brinp1 −/− knock-out mice. a Habituation trial of the three-chamber social interaction test, showing interaction time between empty cages. A significant effect of genotype was observed by one-way ANOVA for male Brinp1 −/− mice only. Female: F(3,19) = 1.482, p = 0.257, male: F(3,19) = 3.428, p = 0.029. b Brinp1 −/− mice show reduced interaction time with a sex-matched novel mouse. A significant effect of genotype was observed by one-way ANOVA; female: F(3,19) = 7.542, p = 0.002, male: F(3,19) = 15.07, p < 0.0001. A Tukey HSD post hoc test showed significant differences between the intruder mouse and empty cage for WT but not Brinp1 −/− mice. Female: WT empty – WT stranger: p = 0.004, Brinp1 −/− empty – Brinp1 −/− stranger: p = 0.199, WT empty – Brinp1 −/− empty: p = 0.996, WT stranger – Brinp1 −/− stranger: p = 0.304, WT empty – Brinp1 −/− stranger: p = 0.136, WT stranger – Brinp1 −/− empty: p = 0.006. Male: WT empty – WT stranger: p < 0.001, Brinp1 −/− empty – Brinp1 −/− stranger: p = 0.528, WT empty – Brinp1 −/− empty: p = 0.910, WT stranger – Brinp1 −/− stranger: p = 0.01, WT empty – Brinp1 −/− stranger: p = 0.887, WT stranger – Brinp1 −/− empty: p < 0.001. c Brinp1 −/− mice exhibited hyperactivity in both trials of three-chamber social interaction test; female: F(1,8) = 55.69, p < 0.001, male: F(1,8) = 41.08, p < 0.001, repeat measures two-way ANOVA. d Ultrasonic vocalisation (USV) of adult male mice: Number of calls, divided into 1 min bins, F(1,17) = 1.693, p = 0.21, repeat measures two-way ANOVA. e Latency of male Brinp1 −/− mice to investigate cotton bud (nose <1 cm from bud), t(17) = 1.835, p = 0.084, and latency to call t(17) = 0.9709, p = 0.3452, unpaired Student’s t tests. f Peak call frequency of USVs from male Brinp1 −/− mice, divided into 1 min bins. No significant effect of genotype was observed: F(1,17) = 3.39, p = 0.089, repeat measures two-way ANOVA. g Male Brinp1 −/− mice emit shorter USV calls. Call duration data was divided into 1 min bins. F(1,17) = 8.17, p = 0.014, repeat measures ANOVA. h Pie chart showing distribution of call types of male Brinp1 −/− mice. For designation of call-type categories, refer to Scattoni et al. [42]. Unidentified calls (11.7 % WT, 11.3 % Brinp1 −/−) were excluded. i Representative spectrogram of first calls from a WT and a male Brinp1 −/− mouse. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001. N = 6 females, 6 males per genotype for sociability experiment (A-C). N = 10 WT, 9 Brinp1-/- male mice for USV experiment (D-I). All data represented as the mean ± SD
Fig. 5
Fig. 5
Brinp1 knock-out mice exhibit hyperactivity and altered exploratory behaviour. a Increased locomotor activity of Brinp1 −/− mice in a 30 min trial in a locomotor cell, shown as a representative data trace for male WT and Brinp1 −/− litter mates. b Increased velocity of Brinp1 −/− mice in the locomotor cell; female: F(1,8) = 27.00, p < 0.001, male: F(1,8) = 8.25, p = 0.021, repeat measures two-way ANOVA. c Increased exploratory behaviour of Brinp1 −/− mice in a locomotor cell, shown as increased time in the centre and reduced time in marginal areas; female: t(8) = 2.919, p = 0.0193, male: t(8)=5.043, p = 0.0054, unpaired Student’s t tests. d Brinp1 −/− mice showed increased stereotypic episodes in the locomotor cells; female: t(8) = 3.414, p = 0.0092, male: t(8) = 3.772, p < 0.0001, unpaired Student’s t tests. e Brinp1 −/− mice exhibited increased rearing behaviour, measured as number of vertical plane (VP) entries; female: t(8) = 2.630, p = 0.0302, male: t(8) = 2.662, p = 0.0287. f Elevated plus maze: male Brinp1 −/− mice spent more time in the open arms relative to the closed arms of the maze, indicating reduced anxiety. Analysis by repeat measures ANOVA shows a genotype × arm interaction for male mice: F(2,20) = 3.525, p = 0.49. Female genotype × arm interaction: F(2,20) = 1.417, p = 0.266. g Brinp1 −/− mice self-directed behaviour: Brinp1 −/− mice were observed for self-directed behaviour in a test plexiglass cage lined with sawdust for 20 min. Male Brinp1 −/− mice showed a decrease in time digging: t(8) = 5.765, p = 0.0004, whilst female Brinp1 −/− mice exhibited decreased grooming duration t(8) = 3.977, p = 0.0041, unpaired Student’s t tests. h WT and Brinp1 −/− mice locomotor activity following injection of MPH/saline in the locomotor cell. An acute IP injection of 2.5 mg/kg of MPH increased locomotor activity for both WT and Brinp1 −/− mice, N = 10 WT MPH, 10 Brinp1 −/− MPH, 10 WT saline, 10 Brinp1 −/− saline. Repeat measures two-way ANOVA analysis, performed on number of distance travelled post injection (20 min+) revealed an effect of genotype; female: F(1,16) = 22.427, p < 0.001, male: F(1,16) = 19.922, p < 0.001, and an effect of 2.5 mg/kg drug treatment; female: F(1,16) = 13.962, p = 0.002, male F(1,16) = 19.239, p < 0.001, whereby administration of MPH resulted in a significant increase in distance travelled for both WT and Brinp1 −/− mice of both sexes. i A dose of 2.5 mg/kg MPH did not significantly alter the number of rearing episodes of Brinp1 −/− mice post drug administration; female: F(3,16) = 0.5698, p = 0.3961, male: F(3, 16) = 0.1351, p = 0.2403, one-way ANOVA. N = 5 female, 5 male mice per genotype for locomotor cell (A-E) and digging/grooming (G) experiments. N = 6 female, 6 male mice per genotype for EPM experiment (F). *P<0.05, **P<0.01, ***P<0.001,****P<0.0001
Fig. 6
Fig. 6
Brinp1 knock-out mice show normal PPI and impaired short-term memory. a. Brinp1 −/− mice showed i) normal startle response; female: t(10) = 0.288, p = 0.779, male: t(10) = 1.045, p = 0.321, unpaired Student’s t test, and ii) and no significant effect of genotype on pre-pulse inhibition (PPI), female: F(1,10) = 0.695, p = 0.424, male: F(1,10) = 0.003, p = 0.958, repeat measures two-way ANOVA. N = 6 female, 6 male mice per genotype. b Y-maze: Results from two cohorts tested indicate that Brinp1−/− mice did not display an increase in time spent exploring the novel arm, in comparison to WT controls. Analysis by repeat measures two-way ANOVA revealed a significant interaction effect for genotype × arm; female: F(2,40) = 3.829, p = 0.030, male: F(2,40) = 3.737, p = 0.033. N = 11 female, 11 male mice per genotype. Data presented as the mean ± SE. c Brinp1 −/− mice showed no impairment in learning the location of a hidden platform in the Morris water maze: i) no significant main effect of genotype on time locating the platform; female: F(1,9) = 0.530, p = 0.819, male: F(1,9) = 0.160, p = 692. N = 5 female, 5 male mice per genotype. Data presented as the mean ± SE. ii) Male Brinp1 −/− mice showed an improvement in locating the platform on day 1, as a decrease in time to find the platform, t(38) = 2.549, p = 0.0173, unpaired Student’s t tests. Data presented as the mean ± SD except where otherwise stated. *p < 0.05, **p < 0.0.1, ***p < 0.001, ****p < 0.0001
Fig. 7
Fig. 7
Increased PV+ interneuron cell density in the adult Brinp1 knock-out neocortex and hippocampus. a Representative coronal section showing parvalbumin (PV)-positive interneurons in the neocortex and medial hippocampus of WT and Brinp1 −/− mice. b An increase in number of PV-positive cells in bins 3 and 5, corresponding with layers IV–VI of the somatosensory neocortex of Brinp1 −/− mice, F(6) = 21.602, p = 0.004, repeat measures two-way ANOVA. c An increase in total numbers of PV-positive cells was observed in the somatosensory neocortex of Brinp1 −/− mice, t(6) = 4.684, p = 0.0034. d PV-positive interneurons in the hippocampus: i) an increase in total numbers of PV-positive cells in the hippocampus of Brinp1 −/− mice t(6) = 2.497, p = 0.0467. ii) A near significant difference was observed for the CA1 region, p = 0.087, whilst no significant differences were observed for CA2, CA3 or dentate gyrus regions. N = 4 mice per genotype
Fig. 8
Fig. 8
Normal cell proliferation in the E18.5 Brinp1 knock-out mouse brain. a No significant change in number of Ki67+ cells (proliferation marker) in the embryonic day 18.5 (E18.5) somatosensory neocortex (coronal) of Brinp1 −/− mice. Neocortex: t(8) = 1.768, p = 0.1150, neocortex SVZ: t(8) = 0.2736, p = 0.7913, unpaired Student’s t tests. N = 5 WT and 5 Brinp1 −/− mice. b No significant change in number of Phh3+ cells (a marker of cells undergoing mitosis) in the neocortex or hippocampus of E18.5 Brinp1 −/− brains (coronal sections). Neocortex SVZ: t(6) = 0.1509, p = 0.8850, neocortex: t(6) = 0.04147, p = 0.9683, hippocampus SVZ: t(6) = 0.1211, p = 0.9083, hippocampus: t(6) = 2.008, p = 0.0914, unpaired Student’s t tests. N = 4 WT and 4 Brinp1 −/− mice. c No significant change in number of activated caspase 3-positive cells (a marker of cells undergoing apoptosis) in the neocortex or hippocampus of E18.5 Brinp1 −/− brain (coronal). Neocortex: t(7) = 0.2564, p = 0.8050, hippocampus: t(7) = 1.114, p = 0.3020, unpaired Student’s t tests. N = 5 WT and 4 Brinp1 −/−mice. d BrdU+ cell distribution in the E18.5 dentate gyrus following administration of BrdU to Brinp1 heterozygous dams at the following time points: embryonic day 12.5 (E12.5), day 14.5 (E14.5) or day 16.5 (E16.5). No significant change in cell number of E12.5-, E14.5- or E16.5-born cells (BrdU-labelled) in the E18.5 dentate gyrus. E12.5 BrdU+ cell count: t(8) = 0.7758, p = 0.4602, E14.5 BrdU+ cell count: t(8) = 0.2668, p = 0.7973, E16.5 BrdU+ cell count: t(8) = 0.01437, p = 0.9890, unpaired Student’s t test. N = 5 WT and 5 Brinp1 −/− mice. All images counter stained with 4′,6-diamidino-2-phenylindole (DAPI, blue)
Fig. 9
Fig. 9
Up-regulation of Astrotactin 1 and Astrotactin 2 mRNA in the embryonic brain and adult hippocampus of Brinp1 knock-out mice. a qPCR showing a significant increase in Astn1 mRNA, t(9) = 2.800, p = 0.0207, and Astn2 mRNA, t(9) = 2.829, p = 0.0222, unpaired Student’s t test, in the developing (E18.5) mouse brain. Levels of Brinp1 exon 3-deleted (Brinp1Δe3) mRNA also increase, compared to levels of Brinp1 in the WT mice: t(9) = 2.733, p = 0.0231. No significant changes overserved for Brinp2 mRNA levels t(9) = 0.4109, p = 0.6908, or Brinp3 mRNA levels: t(9) = 0.5277, p = 0.6105, unpaired Student’s t test. b An increase in Astn1 and Astn2 expression was also detectable in the hippocampus at 6 weeks of the Brinp1 −/− mice: Astn1: t (9) = 3.384, p = 0.0081, Astn2: t(9) = 2.821, p = 0.0200, unpaired Student’s t tests. No significant changes were detected in levels of exon 3-deleted (Brinp1Δe3) mRNA t(9) = 0.8505, p = 0.4171, Brinp2 mRNA t(9) = 1.616, p = 0.1405, or Brinp3 mRNA t(9) = 1.047, p = 0.3222, unpaired Student’s t tests. c In the 6-week-old Brinp1 −/− cortex, less Brinp1 exon 3-deleted (Brinp1Δe3) mRNA (t(5) = 3.611, p = 0.0154) and Brinp2 mRNA (t(5) = 3.113, p = 0.0265) were detected, with no significant change in expression of either Astn1, Astn2 or Brinp3 Astn1: t(5) = 1.572, p = 0.1909, Astn2: t(5) = 1.139, p = 0.3182, Brinp3: t(5) = 1.133, p = 0.3087, unpaired Student’s t tests. N = 3 WT, 4 Brinp1 −/−, *p < 0.05, **p < 0.01. Normalisation against GAPDH expression levels. All data represented as the mean ± SE
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References

    1. Iossifov I, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515(7526):216–21. doi: 10.1038/nature13908. - DOI - PMC - PubMed
    1. Sebat J, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316(5823):445–9. doi: 10.1126/science.1138659. - DOI - PMC - PubMed
    1. Weiss LA. Autism genetics: emerging data from genome-wide copy-number and single nucleotide polymorphism scans. Expert Rev Mol Diagn. 2009;9(8):795–803. doi: 10.1586/erm.09.59. - DOI - PubMed
    1. Folstein SE, Rosen-Sheidley B. Genetics of autism: complex aetiology for a heterogeneous disorder. Nat Rev Genet. 2001;2(12):943–55. doi: 10.1038/35103559. - DOI - PubMed
    1. Fombonne E. Epidemiology of autistic disorder and other pervasive developmental disorders. J Clin Psychiatry. 2005;66(Suppl 10):3–8. - PubMed

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