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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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Light Immunomicroscopy of Pallidin in Mouse Hippocampus.Images A, B, H-K’ depict sections from mouse hippocampus. Sections correspond to immunoperoxidase microscopy with a pallidin monoclonal antibody in wild type (Bloc1s6+/+, A or Mecp2+/y, H), pallidin  (Bloc1s6pa/pa, B) and Mecp2 mutant hippocampi (Mecp2tm1.1Jae/y, I). J–K’ depict indirect immunofluorescence microscopy of Pallidin and VAMP2. Quantitative imaging was performed by confocal microscopy of wild type (Mecp2+/y, J–J′) and Mecp2 mutant hippocampus (Mecp2tm1.1Jae/y, K, K′). VAMP2 was used as a control to normalize staining between animals and experiments. L–M) Box plots depict relative fluorescence intensity expressed as a ratio between pallidin and VAMP2 and the total VAMP2 fluorescence intensity in the dentate gyrus, respectively. Wild type (Mecp2+/y, Blue, n = 4) and Mecp2 mutant tissue (Mecp2tm1.1Jae/y, Red, n = 4) were analyzed. P values were obtained by Mann-Whitney U test. The VAMP2 content is similar between genotypes. Scale bars A = 0.5 mm, H = 1 mm, J′ = 25 µm. DGh, dentate gyrus hilus. Images C–G correspond to sections from human hippocampal tissue stained with pallidin antibody. In C, HF denotes the hippocampal formation, consisting of the hippocampus proper (CA1–3), the dentate gyrus (DG), and the subiculum (Sub). The inner molecular layer of the DG (C, DGiml), the DG hilus (DGh) and the dentat gyrus granule cells layer (DGg) are indicated. Pallidin immunoreactivity is present in cell bodies of CA3 cells (D), subicular pyramidal cells (E), dentate gyrus hilus (F), and dentate gyrus granule cells (G). The presence of pallidin in ectopic granule cells (arrow heads in G) verifies that granule cells, as opposed to terminal fields among them, contain the protein. Note that pallidin is a cytoplasmic, not a nuclear protein. The scale bar in C is 1 mm; that in G is 50 µm.
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pone-0065069-g004: Light Immunomicroscopy of Pallidin in Mouse Hippocampus.Images A, B, H-K’ depict sections from mouse hippocampus. Sections correspond to immunoperoxidase microscopy with a pallidin monoclonal antibody in wild type (Bloc1s6+/+, A or Mecp2+/y, H), pallidin (Bloc1s6pa/pa, B) and Mecp2 mutant hippocampi (Mecp2tm1.1Jae/y, I). J–K’ depict indirect immunofluorescence microscopy of Pallidin and VAMP2. Quantitative imaging was performed by confocal microscopy of wild type (Mecp2+/y, J–J′) and Mecp2 mutant hippocampus (Mecp2tm1.1Jae/y, K, K′). VAMP2 was used as a control to normalize staining between animals and experiments. L–M) Box plots depict relative fluorescence intensity expressed as a ratio between pallidin and VAMP2 and the total VAMP2 fluorescence intensity in the dentate gyrus, respectively. Wild type (Mecp2+/y, Blue, n = 4) and Mecp2 mutant tissue (Mecp2tm1.1Jae/y, Red, n = 4) were analyzed. P values were obtained by Mann-Whitney U test. The VAMP2 content is similar between genotypes. Scale bars A = 0.5 mm, H = 1 mm, J′ = 25 µm. DGh, dentate gyrus hilus. Images C–G correspond to sections from human hippocampal tissue stained with pallidin antibody. In C, HF denotes the hippocampal formation, consisting of the hippocampus proper (CA1–3), the dentate gyrus (DG), and the subiculum (Sub). The inner molecular layer of the DG (C, DGiml), the DG hilus (DGh) and the dentat gyrus granule cells layer (DGg) are indicated. Pallidin immunoreactivity is present in cell bodies of CA3 cells (D), subicular pyramidal cells (E), dentate gyrus hilus (F), and dentate gyrus granule cells (G). The presence of pallidin in ectopic granule cells (arrow heads in G) verifies that granule cells, as opposed to terminal fields among them, contain the protein. Note that pallidin is a cytoplasmic, not a nuclear protein. The scale bar in C is 1 mm; that in G is 50 µm.

Mentions: In order to assess the consequences of a Mecp2 mutation upon pallidin hippocampal protein expression, we first determined the anatomical localization of pallidin, which has not been reported in the mammalian brain. To this end, we used a monoclonal antibody against pallidin in adult mouse and human hippocampal tissue (Fig. 4) [61]. We established the specificity of this antibody in tissue sections from wild-type and pallidin- Bloc1s6pa/pa brain, as demonstrated by the loss of immuno-peroxidase labeling in pallidin- brain (Fig. 4, compare A–B). We then examined the distribution of pallidin in human hippocampal tissue. Strong pallidin immunoreactivity was observed throughout the neuropil of the human hippocampal formation (Fig. 4C, HF). Similar to the distribution of dysbindin we previously reported [33], the highest neuropil levels of pallidin were found in axon terminal fields of glutamatergic neurons intrinsic to the hippocampal formation, most conspicuously in the inner molecular layer of the dentate gyrus (Fig. 4C, DGiml) and among CA2 and CA3 pyramidal cells (Fig. 4C). Figures 4D–G also show that neurons producing these axon terminal fields (hippocampal pyramidal cells in CA3 and polymorph cells in the dentate gyrus hilus [DGh]) are also rich in pallidin (Fig. 4 D, F), as are pyramidal cells in the subiculum (Fig. 4E). Pallidin in dentate gyrus granule cells are clearly seen in Fig. 4G. The distribution of pallidin in mouse brain was similar to the human hippocampus, with a prominent staining in the neuropil of the dentate gyrus hilus and synaptic fields such as the molecular layer of the dentate gyrus (Fig. 4A and H).


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)

Light Immunomicroscopy of Pallidin in Mouse Hippocampus.Images A, B, H-K’ depict sections from mouse hippocampus. Sections correspond to immunoperoxidase microscopy with a pallidin monoclonal antibody in wild type (Bloc1s6+/+, A or Mecp2+/y, H), pallidin  (Bloc1s6pa/pa, B) and Mecp2 mutant hippocampi (Mecp2tm1.1Jae/y, I). J–K’ depict indirect immunofluorescence microscopy of Pallidin and VAMP2. Quantitative imaging was performed by confocal microscopy of wild type (Mecp2+/y, J–J′) and Mecp2 mutant hippocampus (Mecp2tm1.1Jae/y, K, K′). VAMP2 was used as a control to normalize staining between animals and experiments. L–M) Box plots depict relative fluorescence intensity expressed as a ratio between pallidin and VAMP2 and the total VAMP2 fluorescence intensity in the dentate gyrus, respectively. Wild type (Mecp2+/y, Blue, n = 4) and Mecp2 mutant tissue (Mecp2tm1.1Jae/y, Red, n = 4) were analyzed. P values were obtained by Mann-Whitney U test. The VAMP2 content is similar between genotypes. Scale bars A = 0.5 mm, H = 1 mm, J′ = 25 µm. DGh, dentate gyrus hilus. Images C–G correspond to sections from human hippocampal tissue stained with pallidin antibody. In C, HF denotes the hippocampal formation, consisting of the hippocampus proper (CA1–3), the dentate gyrus (DG), and the subiculum (Sub). The inner molecular layer of the DG (C, DGiml), the DG hilus (DGh) and the dentat gyrus granule cells layer (DGg) are indicated. Pallidin immunoreactivity is present in cell bodies of CA3 cells (D), subicular pyramidal cells (E), dentate gyrus hilus (F), and dentate gyrus granule cells (G). The presence of pallidin in ectopic granule cells (arrow heads in G) verifies that granule cells, as opposed to terminal fields among them, contain the protein. Note that pallidin is a cytoplasmic, not a nuclear protein. The scale bar in C is 1 mm; that in G is 50 µm.
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Related In: Results  -  Collection

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

pone-0065069-g004: Light Immunomicroscopy of Pallidin in Mouse Hippocampus.Images A, B, H-K’ depict sections from mouse hippocampus. Sections correspond to immunoperoxidase microscopy with a pallidin monoclonal antibody in wild type (Bloc1s6+/+, A or Mecp2+/y, H), pallidin (Bloc1s6pa/pa, B) and Mecp2 mutant hippocampi (Mecp2tm1.1Jae/y, I). J–K’ depict indirect immunofluorescence microscopy of Pallidin and VAMP2. Quantitative imaging was performed by confocal microscopy of wild type (Mecp2+/y, J–J′) and Mecp2 mutant hippocampus (Mecp2tm1.1Jae/y, K, K′). VAMP2 was used as a control to normalize staining between animals and experiments. L–M) Box plots depict relative fluorescence intensity expressed as a ratio between pallidin and VAMP2 and the total VAMP2 fluorescence intensity in the dentate gyrus, respectively. Wild type (Mecp2+/y, Blue, n = 4) and Mecp2 mutant tissue (Mecp2tm1.1Jae/y, Red, n = 4) were analyzed. P values were obtained by Mann-Whitney U test. The VAMP2 content is similar between genotypes. Scale bars A = 0.5 mm, H = 1 mm, J′ = 25 µm. DGh, dentate gyrus hilus. Images C–G correspond to sections from human hippocampal tissue stained with pallidin antibody. In C, HF denotes the hippocampal formation, consisting of the hippocampus proper (CA1–3), the dentate gyrus (DG), and the subiculum (Sub). The inner molecular layer of the DG (C, DGiml), the DG hilus (DGh) and the dentat gyrus granule cells layer (DGg) are indicated. Pallidin immunoreactivity is present in cell bodies of CA3 cells (D), subicular pyramidal cells (E), dentate gyrus hilus (F), and dentate gyrus granule cells (G). The presence of pallidin in ectopic granule cells (arrow heads in G) verifies that granule cells, as opposed to terminal fields among them, contain the protein. Note that pallidin is a cytoplasmic, not a nuclear protein. The scale bar in C is 1 mm; that in G is 50 µm.
Mentions: In order to assess the consequences of a Mecp2 mutation upon pallidin hippocampal protein expression, we first determined the anatomical localization of pallidin, which has not been reported in the mammalian brain. To this end, we used a monoclonal antibody against pallidin in adult mouse and human hippocampal tissue (Fig. 4) [61]. We established the specificity of this antibody in tissue sections from wild-type and pallidin- Bloc1s6pa/pa brain, as demonstrated by the loss of immuno-peroxidase labeling in pallidin- brain (Fig. 4, compare A–B). We then examined the distribution of pallidin in human hippocampal tissue. Strong pallidin immunoreactivity was observed throughout the neuropil of the human hippocampal formation (Fig. 4C, HF). Similar to the distribution of dysbindin we previously reported [33], the highest neuropil levels of pallidin were found in axon terminal fields of glutamatergic neurons intrinsic to the hippocampal formation, most conspicuously in the inner molecular layer of the dentate gyrus (Fig. 4C, DGiml) and among CA2 and CA3 pyramidal cells (Fig. 4C). Figures 4D–G also show that neurons producing these axon terminal fields (hippocampal pyramidal cells in CA3 and polymorph cells in the dentate gyrus hilus [DGh]) are also rich in pallidin (Fig. 4 D, F), as are pyramidal cells in the subiculum (Fig. 4E). Pallidin in dentate gyrus granule cells are clearly seen in Fig. 4G. The distribution of pallidin in mouse brain was similar to the human hippocampus, with a prominent staining in the neuropil of the dentate gyrus hilus and synaptic fields such as the molecular layer of the dentate gyrus (Fig. 4A and H).

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