Manipulation of neuron identity towards in-vivo circuit reprogramming in the cerebral cortex

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Project Summary Sub-Cerebral Projection Neurons (SCPNs) are a clinically relevant neuron class that controls voluntary movement and whose loss in Amyotrophic Lateral Sclerosis and Fronto-temporal dementia or injury (e.g., damaged by spinal cord injury) leads to paralysis. Currently, there are no methods to replenish injured or lesioned SCPNs. A milestone in regenerative medicine is to utilize cellular reprogramming for brain and circuit repair. This approach uses developmental genes and identity maintenance pathways to switch one cell type to another to replenish vulnerable neuron types. However, there is a significant gap in knowledge in understanding the mechanisms that control identity maintenance in neurons as the brain matures. Our current knowledge is limited to reprogramming projection neurons in-vivo during embryonic stages, limiting potential therapies and the advancement of in-vitro assays. This proposal directly addresses these needs by targeting a identity maintenance mechanism in Layer 6-Cortico Thalamic Neurons (CTns) to shift the balance of cell identity toward Layer 5 (L5) SCPNs. CTns share a developmental origin, differentiation pathways, and morphological characteristics with SCPNs, similarities that are known to facilitate the conversion between cell types. Our group and others have found that CTns can be switched into SCPNs upon loss of the transcriptional regulator Bce1. I hypothesize that knock-out of Bce1 is an effective means of generating SCPNs until post-natal day 14 using re- wiring the CTn axon to the brainstem and the upregulation of L5 markers as readouts of reprogramming success. To target CTns in the motor cortex for reprogramming across developmental stages, I first sought to characterize a novel and broadly necessary Cre- transgenic mouse line (Syt6-Cre) (Aim 1 Part 1). This approach allows access to previously inaccessible CTn populations in the motor areas. Next (Aim 1 Part 2), I will determine the potential and effectiveness of Bce1 mutation in CTns at different maturation stages in generating SCPNs in the motor cortex. In Aim 1 Experiment 1, I investigate the extent that Bce1 mutation induces in-vivo reprogramming of CTns into SCPNs at different cortical maturation stages. I use circuit mapping with retrograde tracers, testing for a new brainstem-projecting axon, and histology for SCPN and CTns molecular markers to determine the efficacy of the approach. Then, in Aim 1 Experiment 2, I use RNA-seq to identify downstream effectors of Bce1 required for reprogrammed CTns to acquire SCPN characteristics. For my (K00 phase), I will transition into the field of CNS repair and regenerative medicine. Aim 2 focuses on visualizing cellular repair within single neurons at the sub-cellular level. This will involve 1) learning a mouse model for CNS injury and repair. Then 2) apply Spatial Transcriptomics to the system. Then 3) test functional hypotheses with high-resolution multi-photon or light sheet microscopy to visualize the process in-vivo. Overall, these techniques will combine my refined skills in light microscopy and developing skills in molecular biology with my interest in CNS repair and are universally applicable thought my career on my path towards independence and a tenure track position.