MeCP2 Regulates the Synaptic Expression of a Dysbindin-BLOC-1 Network Component in Mouse Brain and Human Induced Pluripotent Stem Cell-Derived Neurons

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.Fil: Larimore, Jennifer. Agnes Scott College; Estados UnidosFil: Ryder, Pearl V.. University of Emory; Estados UnidosFil: Kim, Kun Yong. University of Yale. School of Medicine; Estados UnidosFil: Ambrose, L. Alex. Agnes Scott College; Estados UnidosFil: Chapleau, Christopher. University Of Alabama; Estados UnidosFil: Calfa, Gaston Diego. University Of Alabama; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gross, Christina. University of Emory; Estados UnidosFil: Bassell, Gary J.. University of Emory; Estados UnidosFil: Pozzo Miller, Lucas. University Of Alabama; Estados UnidosFil: Smith, Yoland. University of Emory; Estados UnidosFil: Talbot, Konrad. The Pennsylvania State University; Estados UnidosFil: Park, In Hyun. University of Yale. School of Medicine; Estados UnidosFil: Faundez, Victor. University of Emory; Estados Unido

Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism

The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation

Kentucky Outreach Service Kiosk (KyOSK) Study protocol: a community-level, controlled quasi-experimental, type 1 hybrid effectiveness study to assess implementation, effectiveness and cost-effectiveness of a community-tailored harm reduction kiosk on HIV, HCV and overdose risk in rural Appalachia

IntroductionMany rural communities bear a disproportionate share of drug-related harms. Innovative harm reduction service models, such as vending machines or kiosks, can expand access to services that reduce drug-related harms. However, few kiosks operate in the USA, and their implementation, impact and cost-effectiveness have not been adequately evaluated in rural settings. This paper describes the Kentucky Outreach Service Kiosk (KyOSK) Study protocol to test the effectiveness, implementation outcomes and cost-effectiveness of a community-tailored, harm reduction kiosk in reducing HIV, hepatitis C and overdose risk in rural Appalachia.Methods and analysisKyOSK is a community-level, controlled quasi-experimental, non-randomised trial. KyOSK involves two cohorts of people who use drugs, one in an intervention county (n=425) and one in a control county (n=325). People who are 18 years or older, are community-dwelling residents in the target counties and have used drugs to get high in the past 6 months are eligible. The trial compares the effectiveness of a fixed-site, staffed syringe service programme (standard of care) with the standard of care supplemented with a kiosk. The kiosk will contain various harm reduction supplies accessible to participants upon valid code entry, allowing dispensing data to be linked to participant survey data. The kiosk will include a call-back feature that allows participants to select needed services and receive linkage-to-care services from a peer recovery coach. The cohorts complete follow-up surveys every 6 months for 36 months (three preceding kiosk implementation and four post-implementation). The study will test the effectiveness of the kiosk on reducing risk behaviours associated with overdose, HIV and hepatitis C, as well as implementation outcomes and cost-effectiveness.Ethics and disseminationThe University of Kentucky Institutional Review Board approved the protocol. Results will be disseminated in academic conferences and peer-reviewed journals, online and print media, and community meetings.Trial registration numberNCT05657106

<i>Mecp2</i> Deficiency Does Not Affect the Content of BLOC-1 Sensitive Markers in the Dentate Gyrus.

<p>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 <i>Mecp2</i> and BLOC-1 <i>Bloc1s8^sdy/sdy</i>. F–H Box plots depict the quantitation of immunoreactivities of antigens presented in A–E and <i>A–E</i>. P values were obtained by Mann-Whitney U test, n = 4 independent stainings from 2 animals per genotype.</p

Quantitative Real Time PCR Determination of BLOC-1 Subunit Transcripts.

<p>BLOC-1 subunit transcripts from symptomatic adult (A) and P7 (B) mice hippocampi and cortices were analyzed by qRT-PCR. Box plot depicts relative mRNA content for wild type (<i>Mecp2^+/y</i>, Blue) and <i>Mecp2</i> mutant tissue (<i>Mecp2^tm1.1Jae/y</i>, Red). P values were obtained by Mann-Whitney U test. Number of animals tested is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065069#pone-0065069-t001" target="_blank">Table 1</a>.</p

MeCP2 Regulates mRNA Levels of BLOC1-Interacting Proteins.

<p>BLOC1-interacting proteins were compiled from previous reports and mapped using the GeneGo Metacore pathway. The network was visualized in Cytoscope. Node sizes and colors were mapped to mRNA expression level changes in <i>MECP2</i>-overexpressing (A, <i>Mecp2^Tg/y</i>) and <i>Mecp2</i> null mouse (B, <i>Mecp2^−/y</i>) neurons as reported in Chahrour et al., 2008 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065069#pone.0065069-Chahrour2" target="_blank">[29]</a>. We observed that six components of the BLOC1 interactome were modified by Mecp2 gene dosage. The protein products of three of these affected genes, PLDN, COG7, and PRDX1, are decreased in mice null for the schizophrenia susceptibility factor dysbindin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065069#pone.0065069-Gokhale1" target="_blank">[37]</a>.</p

Electron Immunomicroscopy of Pallidin in Mouse Dentate Gyrus.

<p>Images depict immunoperoxidase electron microscopy with a pallidin monoclonal antibody in wild type (<i>Bloc1s6^+/+</i>, A–D or <i>Mecp2^+/y</i>, F–G), pallidin null (<i>Bloc1s6^pa/pa</i>, E) and <i>Mecp2</i> mutant hippocampi (<i>Mecp2^tm1.1Jae/y</i>, H). A–H depict representative asymmetric axospinous synapses from the hilus of the dentate gyrus. <i>Sp</i>, spine; <i>MBV</i>, multivesicular body; <i>CCV</i> clathrin coated vesicle; <i>Cist</i>, cisterna. Structures were identified following defined ultrastructural criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065069#pone.0065069-Peters1" target="_blank">[88]</a>. I) Box plot depicts quantitation of pallidin immuno-positive synaptic compartments in wild type (<i>Bloc1s6^+/+ Mecp2^+/y</i>, Blue, n = 3), pallidin null (<i>Bloc1s6^pa/pa</i>, Red, n = 1) and <i>Mecp2</i> mutant tissue (<i>Mecp2^tm1.1Jae/y</i>, Red, n = 3). Dendrite includes immuno-positive spines as well as dendritic shafts. J) Synapse count and numbers of pallidin immuno-positive synapses per genotype. Bars 200 nm.</p

Quantitative Real Time PCR Determination of Pallidin Transcripts in Neurons derived from Human IPSCs.

<p>(A) Experimental design to generate monoallellic neurons from <i>MECP2</i> mutant Rett syndrome patient cells. (B) Wild type and mutant monoallellic IPSCs from the same patient were differentiated into neurons and analyzed by qRT-PCR. Plot depicts ratio of mRNA content between the wild type and mutant monoallellic cells from the same subject (n = 2 patients). P values were obtained by One Way Anova with Dunnett’s Multiple comparisons correction.</p

Light Immunomicroscopy of Pallidin in Mouse Hippocampus.

<p><i>Images A, B, H-K’ depict sections from mouse hippocampus</i>. Sections correspond to immunoperoxidase microscopy with a pallidin monoclonal antibody in wild type (<i>Bloc1s6^+/+</i>, A or <i>Mecp2^+/y</i>, H), pallidin null (<i>Bloc1s6^pa/pa</i>, B) and <i>Mecp2</i> mutant hippocampi (<i>Mecp2^tm1.1Jae/y</i>, I). J–K’ depict indirect immunofluorescence microscopy of Pallidin and VAMP2. Quantitative imaging was performed by confocal microscopy of wild type (<i>Mecp2^+/y</i>, J–J′) and <i>Mecp2</i> mutant hippocampus (<i>Mecp2^tm1.1Jae/y</i>, 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 (<i>Mecp2^+/y</i>, Blue, n = 4) and <i>Mecp2</i> mutant tissue (<i>Mecp2^tm1.1Jae/y</i>, 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. <i>Images C–G correspond to sections from human hippocampal tissue stained with pallidin antibody</i>. 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.</p

<i>Mecp2</i> and BLOC-1 Deficiency Affect BDNF Immunoreactivity in the Dentate Gyrus.

<p>Images depict indirect quantitative immunofluorescence confocal microscopy of BDNF and the synaptic vesicle marker synaptophysin in the dentate gyrus from <i>Mecp2^tm1.1Jae/y</i>, the BLOC-1-null mice <i>Bloc1s8^sdy/sdy</i>, and <i>Bloc1s6^pa/pa</i> (A–B). BDNF immunorreactivity was abrogated by preincubation of antibodies with recombinant human BDNF (rhBDNF, A–B). Box plots in C depict the quantitation of BDNF immunoreactivity normalized to synaptophysin. P values were obtained by One Way Anova with Dunnett’s Multiple comparisons correction, n = 4 independent stainings from 2 animals per genotype. D, BDNF transcripts from symptomatic adult mice hippocampi were analyzed by qRT-PCR. Box plot depicts relative mRNA content for wild type (<i>Mecp2^+/y</i>, Blue), <i>Mecp2</i> mutant tissue (<i>Mecp2^tm1.1Jae/y</i>, Red), as well as the BLOC-1-null mice <i>Bloc1s8^sdy/sdy</i>, and <i>Bloc1s6^pa/pa</i>. P values were obtained by One Way Anova with Dunnett’s Multiple comparisons correction, n = 4 animals per genotype.</p