Forebrain GABAergic interneurons are the primary source of inhibition in the cerebral cortex and play crucial roles in nearly every aspect of brain function by balancing excitation in cortical circuits. Interneurons comprise approximately 20% of cortical neurons and are divided into different subtypes based on their neurochemical markers, connectivity and physiological properties. However, very little is known about the genetic and molecular mechanisms that give rise to different subtypes of interneuron. The abnormal development and function of cortical interneurons has been implicated in the pathobiology of several major neurological and psychiatric disorders, including schizophrenia, autism, epilepsy, and numerous other diseases. The lack of an efficient mechanism for the production, collection and selection of interneurons has greatly hindered our ability to study both the role of interneurons in disease etiologies, as well as analyze their potential as cell based therapies. The current protocol to derive interneurons from embryonic stem (ES) cells is handicapped by the small percentage of interneurons produced and the biased production of somatostatin- expressing (Sst+) interneurons over other subgroups. In this proposal, we hope to enhance the overall production of ES-derived interneurons and specifically enrich for parvalbumin-expressing (PV+) interneurons. Forced expression of key fate-determining transcription factors can direct the differentiation of stem cells into specific cell types. The transcription factor Nkx2.1 is expressed by cortical interneuron progenitors and is required for the specification of interneuron subtypes. One strategy we are pursuing is to inducibly express Nkx2.1 in ES cells that also contain a post-mitotic reporter for interneuron fate (Lhx6:GFP). Another strategy is to utilize an Nkx2.1:mChery;Lhx6:GFP ES line that allows us to specifically isolate Nkx2.1+ progenitor cells and newly-postmitotic Lhx6+ cels, which could help enrich for PV+ interneurons. We will collect and transplant GFP+ cells into the cortex of neonatal mice to confirm that these cells express neurochemical and electrophysiological properties characteristic of functional interneurons in vivo. After establishing distinct protocols that produce a high percentage of Sst+ and PV+ interneurons, we will collect RNA from GFP+ cells obtained from these different conditions and perform RNA-seq analysis to characterize the transcriptome and identify novel subgroup-specific genes that determine interneuron fate. In sum, the experiments outlined in this proposal should increase our ability to produce ES cell-derived interneurons, and in particular, production of specific subtypes of interneurons. These results will significantly aid the study of interneuron development and advance our capabilities to use interneurons as cell-based therapies for the treatment of disease.
The abnormal development and function of cortical interneurons has been implicated in the pathobiology of several major neurological and psychiatric disorders; including schizophrenia; autism and epilepsy. The lack of an efficient mechanism for the production; collection and selection of interneurons has greatly hindered our ability to study both the role of interneurons in disease etiologies; as well as analyze their potential as cell based therapies. Findings arising from this proposal will greatly enhance our ability to produce interneurons from embryonic stem cells for studying interneuron development and their role in a variety of diseases.
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