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.

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.

Targeting PTEN but not SOCS3 resists an age-dependent decline in promoting axon sprouting.

Axonal repair is critical for functional recovery after injury of the CNS. We previously reported that neuronal PTEN deletion exhibits an age-dependent decline in promoting axon regeneration from the corticospinal tract (CST). How sprouting of uninjured axons, a naturally occurring form of axonal repair, is impacted by age is unknown. We assessed CST sprouting after unilateral pyramidotomy in PTEN and/or SOCS3-deleted mice at different ages. While PTEN deletion enhances sprouting independently of age, SOCS3 deletion loses its sprouting-promoting effect with age. The synergistic effect of PTEN/SOCS3 co-deletion on CST sprouting is rapidly lost with increased age. Overall, promoting sprouting appears more robust across age than regeneration, yet distinct molecular pathways are differentially impacted by age. Importantly, six-week delayed PTEN deletion promotes CST sprouting across age groups, supporting a clinically relevant time frame for this neural repair strategy independently of age.

Overexpressing eukaryotic elongation factor 1 alpha (eEF1A) proteins to promote corticospinal axon repair after injury.

Although protein synthesis is hypothesized to have a pivotal role in axonal repair after central nervous system (CNS) injury, the role of core components of the protein synthesis machinery has not been examined. Notably, some elongation factors possess non-canonical functions that may further impact axonal repair. Here, we examined whether overexpressing eukaryotic elongation factor 1 alpha (eEF1A) proteins enhances the collateral sprouting of corticospinal tract (CST) neurons after unilateral pyramidotomy, along with the underlying molecular mechanisms. We found that overexpressing eEF1A proteins in CST neurons increased the levels of pS6, an indicator for mTOR activity, but not pSTAT3 and pAKT levels, in neuronal somas. Strikingly, overexpressing eEF1A2 alone, but neither eEF1A1 alone nor both factors simultaneously, increased protein synthesis and actin rearrangement in CST neurons. While eEF1A1 overexpression only slightly enhanced CST sprouting after pyramidotomy, eEF1A2 overexpression substantially enhanced this sprouting. Surprisingly, co-overexpression of both eEF1A1 and eEF1A2 led to a sprouting phenotype similar to wild-type controls, suggesting an antagonistic effect of overexpressing both proteins. These data provide the first evidence that overexpressing a core component of the translation machinery, eEF1A2, enhances CST sprouting, likely by a combination of increased protein synthesis, mTOR signaling and actin cytoskeleton rearrangement.

A Critical Role for DLK and LZK in Axonal Repair in the Mammalian Spinal Cord.

The limited ability for axonal repair after spinal cord injury underlies long-term functional impairment. Dual leucine-zipper kinase [DLK; MAP kinase kinase kinase 12; MAP3K12] is an evolutionarily conserved MAP3K implicated in neuronal injury signaling from Caenorhabditis elegans to mammals. However, whether DLK or its close homolog leucine zipper kinase (LZK; MAP3K13) regulates axonal repair in the mammalian spinal cord remains unknown. Here, we assess the role of endogenous DLK and LZK in the regeneration and compensatory sprouting of corticospinal tract (CST) axons in mice of both sexes with genetic analyses in a regeneration competent background provided by PTEN deletion. We found that inducible neuronal deletion of both DLK and LZK, but not either kinase alone, abolishes PTEN deletion-induced regeneration and sprouting of CST axons, and reduces naturally-occurring axon sprouting after injury. Thus, DLK/LZK-mediated injury signaling operates not only in injured neurons to regulate regeneration, but also unexpectedly in uninjured neurons to regulate sprouting. Deleting DLK and LZK does not interfere with PTEN/mTOR signaling, indicating that injury signaling and regenerative competence are independently controlled. Together with our previous study implicating LZK in astrocytic reactivity and scar formation, these data illustrate the multicellular function of this pair of MAP3Ks in both neurons and glia in the injury response of the mammalian spinal cord.SIGNIFICANCE STATEMENT Functional recovery after spinal cord injury is limited because of a lack of axonal repair in the mammalian CNS. Dual leucine-zipper kinase (DLK) and leucine zipper kinase (LZK) are two closely related protein kinases that have emerged as regulators of neuronal responses to injury. However, their role in axonal repair in the mammalian spinal cord has not been described. Here, we show that DLK and LZK together play critical roles in axonal repair in the mammalian spinal cord, validating them as potential targets to promote repair and recovery after spinal cord injury. In addition to regulating axonal regeneration from injured neurons, both kinases also regulate compensatory axonal growth from uninjured neurons, indicating a more pervasive role in CNS repair than originally anticipated.