Dissecting Molecular Mechanisms of Astrocyte-Neuron Interaction in Rett Syndrome Using Arrays of Gene-Edited, Patient-Derived Cells

2017 - presentpresent

Rett syndrome is a pervasive developmental disorder of the brain that dysfunction in both neurons and astrocytes. While neuron-astrocyte non-cell autonomous interaction has been observed in culture and animals, the molecular mechanisms mediating this interaction are poorly understood. We have developed several human stem cell models of Rett syndrome and expertise in astrocyte-neuron coculture as well as morphological and functional analysis of these cells. In addition, we have developed a new platform to array microscale islands of astrocyte-neuron cocultures, gene-edit them in high-throughput, and analyze them in situ with high content analysis without dissociating these cocultures. This novel system effectively separates neurons from dense cultures into isolated ‘islands’ that can be easily analyzed for complexity. Our hypothesis is that automated analysis of spatially isolated astrocyte-neuron cocultures derived from RTT patients will dramatically increase our ability to understand neuronal biology and advance drug discovery/toxicology. We first will test the accuracy and speed of the system when applied to Rett Syndrome (RTT) neural cells that have well-characterized differences in complexity. Next, we will adapt the system for high-throughput analysis of RTT patient-derived neural cells and gene-edits at several candidate genes that may mediate astrocyte-neuronal interaction. If successful, this proposal will establish an innovative approach to measure neuronal complexity using automated methods without requiring manual tracing of neurons, which is labor-intensive and time-consuming. The approach could be applied to a broad spectrum of experiments aimed at understanding the molecular mechanisms of many other neurological disorders, including autism and schizophrenia.