ABSTRACT
Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity that contributes to working-memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal short-term potentiation (STP), a major form of short-term synaptic plasticity. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia.
Subject(s)
DiGeorge Syndrome/genetics , Nuclear Proteins/genetics , Animals , Calcium/metabolism , DiGeorge Syndrome/metabolism , Disease Models, Animal , Haploinsufficiency , Hippocampus/metabolism , Humans , Memory, Short-Term/physiology , Mice , Mitochondria/metabolism , Neuronal Plasticity/genetics , Neuronal Plasticity/physiology , Nuclear Proteins/metabolism , Presynaptic Terminals/metabolism , Ribonucleoproteins , Ribosomal Proteins , Schizophrenia/geneticsABSTRACT
We are taking a cross-species approach to identify genes that are required for mammalian GABAergic neuron differentiation. On the basis of homeodomain similarity, the vertebrate Pitx genes appear to be orthologs of unc-30, a Caenorhabditis elegans gene necessary for differentiation of the GABAergic phenotype of type D neurons. One of the Pitx genes, Pitx2, is expressed in regions of GABAergic neurogenesis in the mammalian brain. These observations led us to test the functional conservation of the mouse Pitx2 and worm unc-30 genes using a rescue assay. Pitx2 rescues the GABAergic differentiation defect and partially rescues the axon guidance and behavioral phenotypes of unc-30 mutants, indicating a high degree of functional conservation between these evolutionarily related genes. Previous studies show that UNC-30 directly regulates the unc-25/glutamate decarboxylase gene that encodes the enzyme for GABA synthesis. We find that the promoter regions of the mouse and human genes coding for the 67 kDa glutamate decarboxylase (Gad1) also contain binding sites matching the UNC-30/Pitx2 consensus binding site sequence. We show that these sites specifically bind to Pitx2 protein in vitro and that in transfected neuroblastoma cells, the Pitx2 binding sites contribute to the basal activity of the Gad1 promoter. Furthermore, in cotransfection experiments, we find that Pitx2 strongly activates the Gad1 promoter. These results indicate that Pitx2 may regulate Gad1 expression in mammals, suggesting a new role for this key developmental transcription factor as a regulator of GABAergic differentiation during mammalian neural development. Our results suggest that some of the mechanisms regulating GABAergic differentiation are evolutionarily conserved.
Subject(s)
Caenorhabditis elegans Proteins , Cell Differentiation/physiology , Helminth Proteins/metabolism , Homeodomain Proteins/metabolism , Neurons/metabolism , Nuclear Proteins , Transcription Factors/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Axons/drug effects , Axons/physiology , Binding Sites/physiology , Caenorhabditis elegans , Cell Differentiation/drug effects , Cell Line , Conserved Sequence/physiology , Gene Expression/drug effects , Genes, Reporter , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/metabolism , Helminth Proteins/genetics , Helminth Proteins/pharmacology , Homeodomain Proteins/genetics , Homeodomain Proteins/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Molecular Sequence Data , Neuroblastoma/metabolism , Neurons/cytology , Neurons/drug effects , Phenotype , Promoter Regions, Genetic/physiology , Protein Structure, Tertiary/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Sequence Homology, Nucleic Acid , Substrate Specificity/genetics , Transcription Factors/genetics , Transcription Factors/pharmacology , Transfection , Transgenes , Homeobox Protein PITX2ABSTRACT
Neural cultures derived from differentiating embryonic stem (ES) cells are a potentially powerful in vitro model of neural development. We show that neural cells derived from mouse ES cells express mRNAs characteristic of GABAergic neurons. The glutamate decarboxylase genes (Gad1 and Gad2), required for GABA synthesis and the vesicular inhibitory amino acid transporter (Viaat) gene, required for GABA vesicular packaging are activated in the ES-derived cultures. Nearly half of the ES-derived neurons express the GAD67 protein, the product of the Gad1 gene. Building on these results we show that Gad1-lacZ "knockin" reporter ES cell lines can be used to easily monitor Gad1 expression patterns and expression levels during ES differentiation. We also demonstrate that the ES-derived neural progenitors can be infected with retroviruses or transfected with plasmids via lipofection. These experiments outline the basic strategies and methods required for studies of GABAergic gene expression and regulation in ES-derived neuronal cultures.