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MeCP2 regulates the synaptic expression of a Dysbindin-BLOC-1 network component in mouse brain and human induced pluripotent stem cell-derived neurons.

Larimore J, Ryder PV, Kim KY, Ambrose LA, Chapleau C, Calfa G, Gross C, Bassell GJ, Pozzo-Miller L, Smith Y, Talbot K, Park IH, Faundez V - PLoS ONE (2013)

Bottom Line: In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients.Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice.Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Agnes Scott College, Decatur, Georgia, USA.

ABSTRACT
Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.

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Mecp2 Deficiency Does Not Affect the Content of BLOC-1 Sensitive Markers in the Dentate Gyrus.Images depict indirect quantitative immunofluorescence microscopy of PI4KIIα and VAMP2 (A–B) or immunoperoxidase light microscopy of VAMP7 (C–D) or the AP-3 δ subunit (E). A–E italic letters represent mutant Mecp2 and BLOC-1 Bloc1s8sdy/sdy. F–H Box plots depict the quantitation of immunoreactivities of antigens presented in A–E and A–E. P values were obtained by Mann-Whitney U test, n = 4 independent stainings from 2 animals per genotype.
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pone-0065069-g006: Mecp2 Deficiency Does Not Affect the Content of BLOC-1 Sensitive Markers in the Dentate Gyrus.Images depict indirect quantitative immunofluorescence microscopy of PI4KIIα and VAMP2 (A–B) or immunoperoxidase light microscopy of VAMP7 (C–D) or the AP-3 δ subunit (E). A–E italic letters represent mutant Mecp2 and BLOC-1 Bloc1s8sdy/sdy. F–H Box plots depict the quantitation of immunoreactivities of antigens presented in A–E and A–E. P values were obtained by Mann-Whitney U test, n = 4 independent stainings from 2 animals per genotype.

Mentions: Reduced pallidin immunoreactivity in Mecp2tm1.1Jae/y dentate gyrus suggests impaired BLOC-1-dependent trafficking in Mecp2tm1.1Jae/y hippocampus. We and others have established that BLOC-1 and its binding partner, the adaptor complex AP-3, generate vesicles that target the SNARE VAMP7 and phosphatidylinositol-4-kinase type IIα (PI4KIIα) to nerve terminals [43]–[45], [65]. Mutations in BLOC-1 subunits lead to reduced AP-3, PI4KIIα, and VAMP7 immunoreactivity in axons and/or asymmetric axospinous nerve terminals in the dentate gyrus [43], [44], [45]. Consistently, AP-3, VAMP7, and PI4KIIα are found in synaptic vesicles [64], [66], [67]. Thus, we tested if Mecp2 mutations would lead to reduced AP-3, PI4KIIα, and/or VAMP7 immunoreactivity in the dentate gyrus secondary to decreased pallidin content. The immunoreactivity of PI4KIIα (Fig. 6A to 6B), VAMP7 (Fig. 6C and C), and the delta subunit of AP-3 (AP-3δ, Fig. 6E and E), were not significantly modified in Mecp2tm1.1Jae/y dentate gyrus. In contrast, VAMP7 immunoreactivity in dysbindin- mice (Bloc1s8sdy/sdy, Fig. 6D–D) was robustly decreased in Bloc1s8sdy/sdy, much like the phenotype previously observed in BLOC-1 mice Bloc1s6pa/pa or Bloc1s5mu/mu[44]. These results suggest that genetic defects in Mecp2 do not affect the delivery of BLOC-1 cargoes to synaptic vesicles.


MeCP2 regulates the synaptic expression of a Dysbindin-BLOC-1 network component in mouse brain and human induced pluripotent stem cell-derived neurons.

Larimore J, Ryder PV, Kim KY, Ambrose LA, Chapleau C, Calfa G, Gross C, Bassell GJ, Pozzo-Miller L, Smith Y, Talbot K, Park IH, Faundez V - PLoS ONE (2013)

Mecp2 Deficiency Does Not Affect the Content of BLOC-1 Sensitive Markers in the Dentate Gyrus.Images depict indirect quantitative immunofluorescence microscopy of PI4KIIα and VAMP2 (A–B) or immunoperoxidase light microscopy of VAMP7 (C–D) or the AP-3 δ subunit (E). A–E italic letters represent mutant Mecp2 and BLOC-1 Bloc1s8sdy/sdy. F–H Box plots depict the quantitation of immunoreactivities of antigens presented in A–E and A–E. P values were obtained by Mann-Whitney U test, n = 4 independent stainings from 2 animals per genotype.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672180&req=5

pone-0065069-g006: Mecp2 Deficiency Does Not Affect the Content of BLOC-1 Sensitive Markers in the Dentate Gyrus.Images depict indirect quantitative immunofluorescence microscopy of PI4KIIα and VAMP2 (A–B) or immunoperoxidase light microscopy of VAMP7 (C–D) or the AP-3 δ subunit (E). A–E italic letters represent mutant Mecp2 and BLOC-1 Bloc1s8sdy/sdy. F–H Box plots depict the quantitation of immunoreactivities of antigens presented in A–E and A–E. P values were obtained by Mann-Whitney U test, n = 4 independent stainings from 2 animals per genotype.
Mentions: Reduced pallidin immunoreactivity in Mecp2tm1.1Jae/y dentate gyrus suggests impaired BLOC-1-dependent trafficking in Mecp2tm1.1Jae/y hippocampus. We and others have established that BLOC-1 and its binding partner, the adaptor complex AP-3, generate vesicles that target the SNARE VAMP7 and phosphatidylinositol-4-kinase type IIα (PI4KIIα) to nerve terminals [43]–[45], [65]. Mutations in BLOC-1 subunits lead to reduced AP-3, PI4KIIα, and VAMP7 immunoreactivity in axons and/or asymmetric axospinous nerve terminals in the dentate gyrus [43], [44], [45]. Consistently, AP-3, VAMP7, and PI4KIIα are found in synaptic vesicles [64], [66], [67]. Thus, we tested if Mecp2 mutations would lead to reduced AP-3, PI4KIIα, and/or VAMP7 immunoreactivity in the dentate gyrus secondary to decreased pallidin content. The immunoreactivity of PI4KIIα (Fig. 6A to 6B), VAMP7 (Fig. 6C and C), and the delta subunit of AP-3 (AP-3δ, Fig. 6E and E), were not significantly modified in Mecp2tm1.1Jae/y dentate gyrus. In contrast, VAMP7 immunoreactivity in dysbindin- mice (Bloc1s8sdy/sdy, Fig. 6D–D) was robustly decreased in Bloc1s8sdy/sdy, much like the phenotype previously observed in BLOC-1 mice Bloc1s6pa/pa or Bloc1s5mu/mu[44]. These results suggest that genetic defects in Mecp2 do not affect the delivery of BLOC-1 cargoes to synaptic vesicles.

Bottom Line: In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients.Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice.Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Agnes Scott College, Decatur, Georgia, USA.

ABSTRACT
Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.

Show MeSH
Related in: MedlinePlus