NOVEL ANIMAL MODELS FOR STUDYING SCHIZOPHRENIA: BAC-DRIVEN MIRNA-MEDIATED IN VIVO SILENCING OF GENE EXPRESSION

Abstract

In schizophrenia, GAD1 disturbances are robust, replicable and represent a core feature of the disease, making it a leading candidate for developing transgenic animal models that mimic the disease phenotype. However, GAD1 downregulation in the prefrontal cortex is not uniform across all the interneuronal subpopulations: parvalbumin- (PARV), somatostatin- (SST) and NPY-containing neurons appear to be preferentially affected in a complex pattern. Importantly, neuropeptide Y (NPY), a phenotypic marker of a subpopulation of GAD1-containing interneurons, has shown reduced expression in the PFC in subjects with schizophrenia, suggesting that dysfunction of the NPY+ cortical interneuronal subpopulation might be also a core feature of this devastating disorder. To mimic these postmortem findings, we generated a transgenic mouse in which we down-regulated GAD1 mRNA expression specifically in NPY+ neurons. This novel, cell type-specific in vivo system for regulation of gene expression utilizes a bacterial artificial chromosome (BAC) containing the NPY promoter-enhancer elements, the reporter molecule (eGFP), and a modified intron containing a synthetic miRNA targeted to GAD1. Furthermore, due to incorporation of an eGFP coding sequence, the targeted cells are identifiable in both live and fixed tissue. The advantages of this in vivo gene silencing system are numerous, and include cell-type specific downregulation of transcripts, visualization of the targeted cells by eGFP, low cost and rapid generation and monoallelic inheritance. Finally, due to the small size of the silencing miRNAs, this method may allow targeting of specific splice-variants, generating splice-variant specific knockdown animals. Combined analysis of the NPY-, PV-, CCK-, and SST-BAC driven, GAD1 miRNA silenced mice (which are currently in production in our laboratory) will greatly contribute to our understanding the mechanisms by which different interneuronal subpopulations mediate cortical inhibition, working memory and cognition. Finally, this knowledge will help us understand the relationship between the cell-type specific GABA-ergic disturbances and the phenotypic manifestations of schizophrenia.