Patch-based deep scRNA-Seq to understand axon repair in the mammalian spinal cord

PROJECT SUMMARY / ABSTRACT Traumatic spinal cord injury is a devastating condition that affect about 302,000 people in the United States, with 18,000 new cases each year. The limited ability of axons to regenerate after injury in the adult central nervous system (CNS) underlies the permanent functional deficits and paralysis experienced by people with spinal cord

injury. Much research remains to be done to fully understand how regeneration is controlled by molecular and cellular machinery in the neurons. This proposal builds on our recent success of applying Patch-based single cell RNA sequencing technology to interrogate the molecular mechanism of corticospinal axon regeneration after

spinal cord injury. By sequencing only hundreds of neurons but at unusually high depths, we developed a regeneration classifier that can be broadly applied to predict the regenerative potential of diverse neuronal types based on their single cell profiles, the first of its kind in regenerative biology. Furthermore, this study implicates

key components in mitochondrial biogenesis and antioxidant response in regulating regeneration. Here we propose to expand Patch-seq based single cell RNA sequencing approach in several ways. First, we will refine and extend the regeneration classifier by sequencing additional corticospinal neurons and other neuronal types

with different regenerative capabilities. This will allow us to develop a more accurate regeneration classifier and understand its full range of capabilities and limitations. Second, we will investigate the role of antioxidant response and mitochondrial biogenesis with a comprehensive array of genetic gain and loss of function analyses

on NFE2L2 and PPARGC1A, master regulators of the two biological processes and two top candidates from our Patch-seq study. Third, we will conduct deep sequencing on young versus old neurons to understand the age impact on axon regeneration, which would be required to develop therapies that are robust across age groups.

Together, the proposed experiments using this unique deep single cell RNA sequencing approach will bring a greater understanding of the neuron intrinsic control of axon regeneration, providing the foundation for therapeutic development to promote repair and recovery after spinal cord injury.