Probing regenerative heterogeneity of corticospinal neurons with scRNA-Seq.

The corticospinal tract (CST) is clinically important for the recovery of motor functions after spinal cord injury. Despite substantial progress in understanding the biology of axon regeneration in the central nervous system (CNS), our ability to promote CST regeneration remains limited. Even with molecular interventions, only a small proportion of CST axons regenerate1. Here we investigate this heterogeneity in the regenerative ability of corticospinal neurons following PTEN and SOCS3 deletion with patch-based single cell RNA sequencing (scRNA-Seq)2,3, which enables deep sequencing of rare regenerating neurons. Bioinformatic analyses highlighted the importance of antioxidant response and mitochondrial biogenesis along with protein translation. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Applying Garnett4, a supervised classification method, to our dataset gave rise to a Regenerating Classifier (RC), which, when applied to published scRNA-Seq data, generates cell type- and developmental stage-appropriate classifications. While embryonic brain, adult dorsal root ganglion and serotonergic neurons are classified as Regenerators, most neurons from adult brain and spinal cord are classified as Non-regenerators. Adult CNS neurons partially revert to a regenerative state soon after injury, which is accelerated by molecular interventions. Our data indicate the existence of universal transcriptomic signatures underlying the regenerative abilities of vastly different neuronal populations, and further illustrate that deep sequencing of only hundreds of phenotypically identified CST neurons has the power to reveal new insights into their regenerative biology.

Deep scRNA sequencing reveals a broadly applicable Regeneration Classifier and implicates antioxidant response in corticospinal axon regeneration.

Despite substantial progress in understanding the biology of axon regeneration in the CNS, our ability to promote regeneration of the clinically important corticospinal tract (CST) after spinal cord injury remains limited. To understand regenerative heterogeneity, we conducted patch-based single-cell RNA sequencing on rare regenerating CST neurons at high depth following PTEN and SOCS3 deletion. Supervised classification with Garnett gave rise to a Regeneration Classifier, which can be broadly applied to predict the regenerative potential of diverse neuronal types across developmental stages or after injury. Network analyses highlighted the importance of antioxidant response and mitochondrial biogenesis. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Our data demonstrate a universal transcriptomic signature underlying the regenerative potential of vastly different neuronal populations and illustrate that deep sequencing of only hundreds of phenotypically identified neurons has the power to advance regenerative biology.