Engineering alternative splicing programs to accelerate maturation of stemcell-derived neurons

Engineering alternative splicing programs to accelerate maturation of stem cell-derived neurons PROJECT SUMMARY Incurable aging-related neurodegenerative diseases are a growing public health crisis. The ability to generate substantial quantities of disease-pertinent neuronal types, with and without predisposing mutations, holds great promise for probing disease mechanisms and developing therapies. However, while tremendous progress has made it possible to differentiate pluripotent stem cells to neurons of various developmental lineages, current protocols yield neurons that fail to mature in vitro and stall at an embryonic identity. This reflects a fundamental gap in knowledge concerning the underlying molecular programs that drive neuronal maturation and aging, limiting the potential of stem-cell-based interrogations of late-onset diseases. This study aims to address this critical conceptual and technological challenge. The mammalian nervous system employs alternative splicing to massively expand the complexity of genetic information encoded in the genome. Our recent studies on developmental alternative splicing programs revealed that thousands of alternative exons show dramatic splicing switches at precise time points during neuronal maturation; these switches lead to expression of adult transcript and protein isoforms involved in morphological and functional maturation of neurons. Importantly, we identified core RNA-binding protein (RBP) splicing factors, whose expression is developmentally regulated to drive the coordinated maturation-dependent splicing changes. These discoveries have led us to hypothesize that engineering alternative splicing programs by manipulating the expression of key splicing factors in nascent neurons will be sufficient to overcome gene regulatory barriers and accelerate neuronal maturation at the molecular, cellular and functional levels, in a similar way that ectopic expression of Yamanaka factors reprograms somatic cells to pluripotent stem cells. In this study, we will combine the strengths of Wichterle lab in spinal motor neuron development and Zhang lab in systems RNA biology to test this hypothesis by focusing on stem cell-derived spinal motor neuron as a model. We will test different combinations of candidate RBPs, timing and duration of heterochronic RBP expression to find the optimal experimental condition that can effectively activate the mature splicing programs. Systematic evaluations will be performed to assess molecular, morphological, and electrophysiological maturation of the reprogrammed cells. If successful, this project will develop a platform technology with a broad impact on studies of the adult and aging nervous system in health and disease.