Human spinal cord injury leads to life-long disability, with limited treatment options despite decades of research in rodents. Recent evidence indicates that there are fundamental differences in gene expression between human and mouse, suggesting a critical need for identification of novel or human-specific candidates as targets to increase axon regeneration following spinal cord injury. Additionally, in vitro studies of axon growth are typically performed in neurons from embryonic or young postnatal rodents, ages with increased intrinsic growth ability, and a very different transcriptional landscape from adult neurons. Thus, we propose to create an age-relevant, human model for axon growth and regeneration. Recent technological advances now allow for human adult somatic cells to be reprogrammed into stem cells, and then differentiated into neurons. However, reprogramming these cells through pluripotency has been shown to erase the ?age? of the original cell, resulting in a more embryonic identity of the resulting neuron. Using a direct reprogramming protocol, we will transdifferentiate neonatal, ~35 year old, and ~70 year old human somatic cells directly into neurons through overexpression of specific neuronal factors, allowing for maintenance of the cell?s biological age identity. We will determine the maintenance of age in these cells at the level of the epigenetic age signature, gene expression, and markers of cellular aging. As the first study of axon growth in age-maintained human neurons, we will characterize the basal axon growth behavior on permissive and inhibitory substrates to identify if age plays a role in human axon growth ability. To test this new resource, we will target 20 different genes and pathways previously identified in other species to regulate axon growth for their conservation in function in human neurons. In rodents it has recently been shown that certain axon growth modifiers such as PTEN knockout, do not function similarly in aged animals as young animals (Geoffroy et al, 2016). Thus, we will utilize our age-maintained human iNs to determine if age drives changes in axon growth phenotypes. Completion of this research will 1) establish a new human, age-relevant cell culture system for axon growth studies, 2) identify genes and their regulators that have conserved functions in human neurons, and 3) determine the role age plays in axon growth ability. Identification of translational targets specifically in human neurons ultimately may lead to the development of effective therapeutic targets for human spinal cord injury.
Despite decades of research in rodents, there are currently limited successful treatments for human spinal cord injury. We propose to develop a human, age-specific neuronal model to study axon growth, determine the conservation of axon growth modulators identified in other species in the human neurons, and reveal what role age may play in human axon growth ability. This research will result in the creation of a powerful resource for the axon growth community, and lead to the development of effective therapeutic targets for human spinal cord injury.
