Molecular regulation over the decline in long-distance corticospinal axon regenerative ability during development - Corticospinal neurons (CSN) reside in the neocortex, and extend axons to specific segmental targets in the spinal cord forming the corticospinal tract (CST). CSN critically control voluntary movement and are centrally involved in recovery from paralysis originating from multiple causes, e.g., stroke, spinal cord injury (SCI), cerebral palsy, etc.. CSN degeneration in amyotrophic lateral sclerosis (ALS), along with degeneration of spinal motor neurons, causes spasticity and paralysis. Recovery in all these distinct causes of paralysis would ultimately require long-distance CST regeneration, which remains an unattained goal in regenerative neuroscience. Previous work using neonatal lesions has established CST regenerative ability declines from development into adulthood. When the CST is damaged in early life, there is greater plasticity and regeneration as compared to similar lesions in the adult. However, despite this work, we still do not know when long-distance regenerative ability is lost during development. This is because a key limitation of these established neonatal lesion models is that they massively disrupt the spinal environment and thereby interfere with the normal process of long- distance CSN axon extension during development. This limits the ability of these lesion paradigms to assess the ability of the CNS to support long-distance regeneration. We recently established a novel microsurgical approach to axotomize the CST during development, while leaving the spinal environment relatively intact. We identified that long-distance CST regenerative ability is lost at different times at distinct spinal levels– at postnatal day 4 (P4) the CST can fully regenerate when lesioned at thoracic T11, but fails to do so when lesioned at cervical C2. Our results indicate that the loss of long-distance CST regenerative ability closely parallels the developmental timeline of normal CST growth into the spinal cord, which suggests that the normal process of differentiation, both in CSN and in the spinal cord, results in the loss of long-distance regenerative ability. This proposal investigates the hypothesis that inhibition of differentiation to prolong the immature developmental state will extend the time window when long-distance regeneration is possible. Specifically, we will manipulate the function of RE1 silencing transcription factor (REST), a global repressor of neural differentiation, to test this hypothesis. Building on this foundation, we will first manipulate REST function (both gain- and loss-of function) at distinct spinal levels to investigate whether this affects the ability of these spinal segments to support long-distance CST regeneration (Aim1). We will manipulate REST function in CSN to similarly investigate long-distance CST regeneration (Aim 2). Finally, we will use single cell profiling to investigate potentially distinct molecular effects of microlesions at distinct spinal levels on distinct CSN subsets depending on their developmental state (Aim 3). Together, our work will discern novel molecular mechanisms of how long-distance CST regeneration is regulated during development, thereby providing a mechanistic framework for subsequent identification of molecules that can be used to effect long-distance CST regeneration in the adult CNS.
