Subcellular investigation of molecular programs responsible for corticospinal neuron development and treatment-enhanced regeneration

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Traumatic spinal cord injury (SCI) is an acquired disorder causing permanent functional deficits due to lack of regenerative ability of the central nervous system (CNS). The inability of the CNS to re-generate is in stark contrast with its ability to generate precise circuitry during development. Corticospinal neurons (CSN) are the subtype of cortical projection neurons (PN) that normally connect the cerebral cortex to the spinal cord to control voluntary motor output directly and indirectly. During development, CSN axons traverse vast distances to establish segmentally-specific functional circuitry along the rostro-caudal spinal cord. Establishment of such specific circuitry necessitates tightly regulated, dynamic developmental programs to progressively refine CSN identity and their input and output connections. After injury, CSN do not normally re-establish functional circuitry. Despite decades of research, and existence of multiple animal models of increased CSN regeneration, the extent of functional recovery for people with SCI remains largely unchanged. This lack of clinical advance is partly due to limitations of understanding of molecular mechanisms directly responsible for locally enacting axon growth and guidance (or not), both during development and during attempted regeneration. In my proposed work, I will investigate the distinct transcriptomes and proteomes of CSN growth cones (GCs) vs. somata during development and after injury, toward selecting molecular candidates for functional manipulation. GCs are the cellular subcompartments at the ends of growing axons that directly enact neuronal subtype- specific axon growth and guidance during development and after injury. Direct investigation of CSN GC molecular machinery promises to elucidate local subcellular processes that underpin developmental and regenerative CSN growth. My lab has recently developed experimental and analytic approaches to deeply investigate subtype- and stage-specific GCs in vivo. These approaches have led to identification of neuronal subtype-specific regulation of local RNA, protein, and translation in interhemispheric callosal and corticothalamic projection neurons. My lab and I have purified thoracolumbar CSN (CSNTL) GCs and somata from postnatal day 3 (P3), P5, and P7 mice, during axon elongation, grey matter innervation, and branching. I will analyze the RNA sequencing data obtained, and will expand by addition of proteomics, to select candidates for functional investigation (Aim 1). I will use a model of increased CSN regeneration after SCI (Pten deletion) to investigate local RNA-protein of regenerating CSNTL (Aim 2). Why CNS regeneration does not occur after injury is a critical unanswered, fundamental question with immense translational implications. My work aims to elucidate developmental growth programs responsible for directing appropriate CSN axon elongation, segment- specific branching and collateralization, and synapse targeting. Importantly, my work also aims to elucidate molecular mechanisms responsible for enabling growth of the CNS after injury. Deeper elucidation of molecular mechanisms of regenerative growth will enable future development of targeted therapies for disability from SCI.