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RBFOX3/NeuN is Required for Hippocampal Circuit Balance and Function.

Wang HY, Hsieh PF, Huang DF, Chin PS, Chou CH, Tung CC, Chen SY, Lee LJ, Gau SS, Huang HS - Sci Rep (2015)

Bottom Line: RBFOX3 mutations are linked to epilepsy and cognitive impairments, but the underlying pathophysiology of these disorders is poorly understood.Focusing on hippocampal phenotypes, we found Rbfox3 knockout mice showed increased expression of plasticity genes Egr4 and Arc, and the synaptic transmission and plasticity were defective in the mutant perforant pathway.The mutant dentate granules cells exhibited an increased frequency, but normal amplitude, of excitatory synaptic events, and this change was associated with an increase in the neurotransmitter release probability and dendritic spine density.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei, 10051, Taiwan.

ABSTRACT
RBFOX3 mutations are linked to epilepsy and cognitive impairments, but the underlying pathophysiology of these disorders is poorly understood. Here we report replication of human symptoms in a mouse model with disrupted Rbfox3. Rbfox3 knockout mice displayed increased seizure susceptibility and decreased anxiety-related behaviors. Focusing on hippocampal phenotypes, we found Rbfox3 knockout mice showed increased expression of plasticity genes Egr4 and Arc, and the synaptic transmission and plasticity were defective in the mutant perforant pathway. The mutant dentate granules cells exhibited an increased frequency, but normal amplitude, of excitatory synaptic events, and this change was associated with an increase in the neurotransmitter release probability and dendritic spine density. Together, our results demonstrate anatomical and functional abnormality in Rbfox3 knockout mice, and may provide mechanistic insights for RBFOX3-related human brain disorders.

No MeSH data available.


Related in: MedlinePlus

Validation and phenotypic characterization of Rbfox3−/− mice.(a) Schematic of the targeting locus on the Rbfox3 gene. A LacZ-Neo cassette was inserted into the intron between exon 6 and 7 and FRT sites flanked the cassette. LoxP sites flanked exons 7 to 9. (b) Genotyping by PCR analysis of tail genomic DNA [WT (+/+), heterozygous (+/−), and homozygous (−/−) mutant] using Rbfox3, LacZ and Neo primers (indicated in a). (c) Quantitative RT-PCR analysis of cerebral cortical Rbfox3 transcripts from WT and Rbfox3 homozygous knockout (Rbfox3−/−) (KO) mice. Student’s t-test, two-tailed **P < 0.01, n = 4 per group. (d) Western blotting analysis of hippocampal RBFOX3 protein from WT and KO mice probed with antibodies to RBFOX3 and ACTIN. Student’s t-test, two-tailed *P < 0.05, n = 4–6 per group. (e) Hippocampal regions from WT and KO mice immunostained with RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (f) Somatosensory cortical regions from WT and KO mice immunostained with anti-RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (g) Body weights and (h) brain weights from WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, ***P < 0.001, n = 7-13 per group. Neuronal integrity determined with western blotting analysis of neurofilament protein heavy chain (NF200-H) (i, left) and its phosphorylated (pNF-H) (i, middle) and non-phosphorylated (NF-H) form (i, right), neurofilament protein light chain (j), neurofilament protein medium chain (k) in the hippocampus of WT and KO mice. Student’s t-test, two-tailed or Mann-Whitney rank sum test *P < 0.05, n = 6-8 per group. (l) White matter volume determined with MRI analysis in the whole brain of WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, n = 6 per group.
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f1: Validation and phenotypic characterization of Rbfox3−/− mice.(a) Schematic of the targeting locus on the Rbfox3 gene. A LacZ-Neo cassette was inserted into the intron between exon 6 and 7 and FRT sites flanked the cassette. LoxP sites flanked exons 7 to 9. (b) Genotyping by PCR analysis of tail genomic DNA [WT (+/+), heterozygous (+/−), and homozygous (−/−) mutant] using Rbfox3, LacZ and Neo primers (indicated in a). (c) Quantitative RT-PCR analysis of cerebral cortical Rbfox3 transcripts from WT and Rbfox3 homozygous knockout (Rbfox3−/−) (KO) mice. Student’s t-test, two-tailed **P < 0.01, n = 4 per group. (d) Western blotting analysis of hippocampal RBFOX3 protein from WT and KO mice probed with antibodies to RBFOX3 and ACTIN. Student’s t-test, two-tailed *P < 0.05, n = 4–6 per group. (e) Hippocampal regions from WT and KO mice immunostained with RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (f) Somatosensory cortical regions from WT and KO mice immunostained with anti-RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (g) Body weights and (h) brain weights from WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, ***P < 0.001, n = 7-13 per group. Neuronal integrity determined with western blotting analysis of neurofilament protein heavy chain (NF200-H) (i, left) and its phosphorylated (pNF-H) (i, middle) and non-phosphorylated (NF-H) form (i, right), neurofilament protein light chain (j), neurofilament protein medium chain (k) in the hippocampus of WT and KO mice. Student’s t-test, two-tailed or Mann-Whitney rank sum test *P < 0.05, n = 6-8 per group. (l) White matter volume determined with MRI analysis in the whole brain of WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, n = 6 per group.

Mentions: To address the potential causal role of RBFOX3 in neurological disorders, we first generated Rbfox3 homozygous knockout (Rbfox3−/−) mice carrying a knockout-first and conditional-ready allele of the Rbfox3 gene (Fig. 1a). We verified the genotype of Rbfox3−/− mice using Rbfox3 primers for the wild-type allele, and LacZ and Neo primers for the mutant Rbfox3 allele (Fig. 1a,b). We observed little evidence of Rbfox3 mRNA in the brains using quantitative real-time PCR (Fig. 1c) or RBFOX3 protein in the hippocampus by western blotting analysis (Fig. 1d) of Rbfox3−/− mice. In addition, no truncated or fusion proteins were generated as a result of the mutation in Rbfox3−/− mice (see Supplementary Fig. S1 online). Immunostaining of tissue sections showed that Rbfox3−/− mice lacked RBFOX3 protein signal in the hippocampus (Fig. 1e) and cerebral cortex (Fig. 1f). The mutation did not seem to cause major defects in brain morphology as DAPI staining on brain tissues appeared grossly normal in Rbfox3−/− mice (Fig. 1e,f)


RBFOX3/NeuN is Required for Hippocampal Circuit Balance and Function.

Wang HY, Hsieh PF, Huang DF, Chin PS, Chou CH, Tung CC, Chen SY, Lee LJ, Gau SS, Huang HS - Sci Rep (2015)

Validation and phenotypic characterization of Rbfox3−/− mice.(a) Schematic of the targeting locus on the Rbfox3 gene. A LacZ-Neo cassette was inserted into the intron between exon 6 and 7 and FRT sites flanked the cassette. LoxP sites flanked exons 7 to 9. (b) Genotyping by PCR analysis of tail genomic DNA [WT (+/+), heterozygous (+/−), and homozygous (−/−) mutant] using Rbfox3, LacZ and Neo primers (indicated in a). (c) Quantitative RT-PCR analysis of cerebral cortical Rbfox3 transcripts from WT and Rbfox3 homozygous knockout (Rbfox3−/−) (KO) mice. Student’s t-test, two-tailed **P < 0.01, n = 4 per group. (d) Western blotting analysis of hippocampal RBFOX3 protein from WT and KO mice probed with antibodies to RBFOX3 and ACTIN. Student’s t-test, two-tailed *P < 0.05, n = 4–6 per group. (e) Hippocampal regions from WT and KO mice immunostained with RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (f) Somatosensory cortical regions from WT and KO mice immunostained with anti-RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (g) Body weights and (h) brain weights from WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, ***P < 0.001, n = 7-13 per group. Neuronal integrity determined with western blotting analysis of neurofilament protein heavy chain (NF200-H) (i, left) and its phosphorylated (pNF-H) (i, middle) and non-phosphorylated (NF-H) form (i, right), neurofilament protein light chain (j), neurofilament protein medium chain (k) in the hippocampus of WT and KO mice. Student’s t-test, two-tailed or Mann-Whitney rank sum test *P < 0.05, n = 6-8 per group. (l) White matter volume determined with MRI analysis in the whole brain of WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, n = 6 per group.
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Related In: Results  -  Collection

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f1: Validation and phenotypic characterization of Rbfox3−/− mice.(a) Schematic of the targeting locus on the Rbfox3 gene. A LacZ-Neo cassette was inserted into the intron between exon 6 and 7 and FRT sites flanked the cassette. LoxP sites flanked exons 7 to 9. (b) Genotyping by PCR analysis of tail genomic DNA [WT (+/+), heterozygous (+/−), and homozygous (−/−) mutant] using Rbfox3, LacZ and Neo primers (indicated in a). (c) Quantitative RT-PCR analysis of cerebral cortical Rbfox3 transcripts from WT and Rbfox3 homozygous knockout (Rbfox3−/−) (KO) mice. Student’s t-test, two-tailed **P < 0.01, n = 4 per group. (d) Western blotting analysis of hippocampal RBFOX3 protein from WT and KO mice probed with antibodies to RBFOX3 and ACTIN. Student’s t-test, two-tailed *P < 0.05, n = 4–6 per group. (e) Hippocampal regions from WT and KO mice immunostained with RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (f) Somatosensory cortical regions from WT and KO mice immunostained with anti-RBFOX3 antibody (green) and counter-stained with DAPI (blue). Scale bar = 200 μm. (g) Body weights and (h) brain weights from WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, ***P < 0.001, n = 7-13 per group. Neuronal integrity determined with western blotting analysis of neurofilament protein heavy chain (NF200-H) (i, left) and its phosphorylated (pNF-H) (i, middle) and non-phosphorylated (NF-H) form (i, right), neurofilament protein light chain (j), neurofilament protein medium chain (k) in the hippocampus of WT and KO mice. Student’s t-test, two-tailed or Mann-Whitney rank sum test *P < 0.05, n = 6-8 per group. (l) White matter volume determined with MRI analysis in the whole brain of WT and KO mice. Student’s t-test, two-tailed, *P < 0.05, n = 6 per group.
Mentions: To address the potential causal role of RBFOX3 in neurological disorders, we first generated Rbfox3 homozygous knockout (Rbfox3−/−) mice carrying a knockout-first and conditional-ready allele of the Rbfox3 gene (Fig. 1a). We verified the genotype of Rbfox3−/− mice using Rbfox3 primers for the wild-type allele, and LacZ and Neo primers for the mutant Rbfox3 allele (Fig. 1a,b). We observed little evidence of Rbfox3 mRNA in the brains using quantitative real-time PCR (Fig. 1c) or RBFOX3 protein in the hippocampus by western blotting analysis (Fig. 1d) of Rbfox3−/− mice. In addition, no truncated or fusion proteins were generated as a result of the mutation in Rbfox3−/− mice (see Supplementary Fig. S1 online). Immunostaining of tissue sections showed that Rbfox3−/− mice lacked RBFOX3 protein signal in the hippocampus (Fig. 1e) and cerebral cortex (Fig. 1f). The mutation did not seem to cause major defects in brain morphology as DAPI staining on brain tissues appeared grossly normal in Rbfox3−/− mice (Fig. 1e,f)

Bottom Line: RBFOX3 mutations are linked to epilepsy and cognitive impairments, but the underlying pathophysiology of these disorders is poorly understood.Focusing on hippocampal phenotypes, we found Rbfox3 knockout mice showed increased expression of plasticity genes Egr4 and Arc, and the synaptic transmission and plasticity were defective in the mutant perforant pathway.The mutant dentate granules cells exhibited an increased frequency, but normal amplitude, of excitatory synaptic events, and this change was associated with an increase in the neurotransmitter release probability and dendritic spine density.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei, 10051, Taiwan.

ABSTRACT
RBFOX3 mutations are linked to epilepsy and cognitive impairments, but the underlying pathophysiology of these disorders is poorly understood. Here we report replication of human symptoms in a mouse model with disrupted Rbfox3. Rbfox3 knockout mice displayed increased seizure susceptibility and decreased anxiety-related behaviors. Focusing on hippocampal phenotypes, we found Rbfox3 knockout mice showed increased expression of plasticity genes Egr4 and Arc, and the synaptic transmission and plasticity were defective in the mutant perforant pathway. The mutant dentate granules cells exhibited an increased frequency, but normal amplitude, of excitatory synaptic events, and this change was associated with an increase in the neurotransmitter release probability and dendritic spine density. Together, our results demonstrate anatomical and functional abnormality in Rbfox3 knockout mice, and may provide mechanistic insights for RBFOX3-related human brain disorders.

No MeSH data available.


Related in: MedlinePlus