Debilitating degenerative disorders like Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis cause progressive loss of adolescent and adult motor neurons. The ability to produce motor neurons in vitro from patient-derived induced pluripotent stem cells holds great promise for mechanistically understanding these diseases and testing drugs for treatment. However, motor neurons differentiated from stem cells using current methods fail to mature, molecularly resembling fetal rather than adult cells. This experimental limitation parallels a lack of understanding of motor neuron maturation in vivo, and prevents the in-depth study of neuronal function and dysfunction in adolescent and adult onset disorders. The goal of this proposal is to define the transcriptional regulators of motor neuron maturation in vivo and to develop methods for programming adult motor neurons in vitro. Expression analyses demonstrate that neuronal maturation is accompanied by global changes in gene expression. In the proposed study, the regulatory logic of maturation-specific gene expression will first be defined by performing genomic analysis on aging mouse motor neurons. Specifically, the recently developed INTACT method will be used to purify embryonic, post-natal, and adult motor neuron nuclei in order to map active enhancers and gene expression. Motif analysis will then be performed at stage-specific enhancers, in combination with differential expression analysis, to computationally identify the transcription factors that regulate maturation. Next, the sufficiency of putative regulatory transcription factors to specify adult motor neurons will be tested in in vitro maturation assays. To do this, stem cell lines with stage-specific reporters will first be created to track maturation stage. These cells will be differentiated into immature motor neurons, expression of putative maturation-regulating transcription factors will be induced, and combinations of transcription factors that lead to the activation of maturation markers will be identified. This approach will not only identify transcription factors that control mature motor neuron identity in vivo, but will also allow the manipulation of transcription factor expression in vitro to produce developmentally relevant, adult motor neurons from pluripotent cells. Overall, the proposed study will illuminate regulatory mechanisms that control maturation of spinal motor neurons, enable further mechanistic studies of neuronal maturation, and make it possible to appropriately model adolescent and adult onset degenerative disorders.
The proposed project will define the transcriptional regulation of post-natal spinal motor neuron maturation and inform methods for accelerated production of adult-like motor neurons from pluripotent cells. Degenerative disorders like Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis specifically affect post-natal motor neurons in adolescent and adult individuals. This work will further our understanding of disease pathologies in such disorders, instruct methods for the production of adult-like motor neurons from patient derived pluripotent cells, allow mechanistic studies of motor neuron function and dysfunction, and facilitate drug screens.
