Recent data indicate that the induction of regenerative gene expression, a prerequisite for activating axon growth programs, relies partly on creating a more permissive chromatin environment in the nucleus of injured neurons ^{45– 51} . Epigenomic screens of adult dorsal root ganglia (DRG) neurons injured by a peripheral nerve lesion (PNL), an experimental condition that switches DRG neurons into a regenerative-competent state, identified Tet methylcytosine dioxygenase 3 (Tet3) as a critical regulator of axon growth and regeneration. After PNL, Tet3 is upregulated along with the epigenetic mark 5-hydroxymethylcytosine (5hmC) in DRG neurons ⁵² . By oxidizing 5hmC, Tet3 reverses DNA methylation. Interestingly, epigenomic mapping in DRG neurons after injury to the peripheral (regenerative) or central (no regenerative effect) projecting axons triggered differential 5hmC changes that were associated with distinct signaling pathways ⁵² . Nearly half of the genes that were differentially regulated after peripheral lesion contain 5hmC alterations ⁵² , suggesting that 5hmC is a previously unrecognized mechanism that controls the regenerative potential of injured neurons.

Although transcriptional events that turn on the expression of regeneration-associated genes are recognized as important steps in the activation of cell-autonomous regeneration programs ⁵³ , far less is known about how gene inactivation affects these programs. A recent study identified ubiquitin-like containing PHD ring finger 1 (UHRF1)-dependent DNA methylation as a critical epigenetic mechanism responsible for silencing expression of genes that are required to promote axon regeneration in DRG neurons ⁵⁴ . After PNL, a decrease in miR-9 causes a transient increase in the expression of the RE1-silencing transcription factor (REST) and UHRF1 ⁵⁴ . During embryogenesis, REST acts as master regulator by inhibiting the expression of many neuronal genes ⁵⁵ . While a transient increase in REST primes injured DRG neurons for enhanced axon regeneration, UHRF1 interacts with DNA methyltransferases and methyl groups on histone H3, creating epigenetic marks that silence promoter elements of tumor suppressor genes such as the phosphatase and tensin homolog ( PTEN) and REST ⁵⁴ . Since sustained expression of REST in neurons is known to cause axon guidance defects ⁵⁶ , UHRF1-dependent epigenetic silencing may be required to fine-tune REST activity and thus axon regeneration programs. Together, these data support the idea that neurons may need to revert to an immature or intermediate state to successfully unlock developmental programs for axon regeneration ^{57– 62} .

Advanced transcriptomics analyses have identified several genes and gene networks that regulate axon regeneration success and failure ^{27, 34, 63, 64} . However, the efficiency and success of translation of these genes have received less attention. By ribosome pull-down and metabolic isotopic labeling, a recent study analyzed gene translation and protein synthesis within the regeneration-associated program in DRG neurons. Of the proteins that undergo de novo synthesis in regenerating DRG neurons, apolipoprotein E (ApoE), which has been previously implicated in axon growth and regeneration ^{65– 68} , is one of the most robustly synthesized proteins ⁶⁹ . DRG neurons cultured in the presence of an ApoE receptor inhibitor extend shorter neurites, providing evidence that neuronal ApoE is an autocrine regulator of axon growth ⁶⁹ . It is likely, though speculative, that ApoE facilitates recycling of cholesterol from degenerating axons for integration into new membranes during the process of axon regeneration. Alternatively, cholesterol may be synthesized in the cell body and then efficiently delivered to the axonal compartment via anterograde transport of lipid-containing vesicles.

An unbiased genome-wide loss-of-function screen in cerebral cortical projection neurons in vitro identified Rab27b, a member of the Rab subfamily of GTPases, as a cell-autonomous factor that restricts axon regeneration ³⁹ . Adult worms lacking Rab27 exhibit greater regeneration of GABA neurons. Moreover, optic nerve regeneration, raphespinal sprouting, and locomotor recovery all are enhanced in mice lacking Rab27 ³⁹ . Interestingly, Caenorhabditis elegans Rab27 mutants have defects in synaptic transmission ⁷⁰ . Given that Rab27 localizes in synaptic-rich regions and participates in the transport of synaptic vesicles ⁷¹ , removing or blocking Rab27 in adult neurons may promote axon regeneration by shifting the trafficking of new cell membrane from synapses to the axolemma. Indeed, new membrane insertion is necessary for axon elongation ⁷² .

Interestingly, data from an independent study show that selective exclusion of Rab11 vesicles, which are needed for axon elongation, contributes to axon regeneration failure. Rab GTPases coordinate vesicle trafficking ⁷³ , thereby allowing growth-promoting cargoes to be delivered to the axon. In cultured rat cortical neurons, overexpressing Rab11 decreases axon retraction and augments new growth cone formation and enhanced axon regeneration occurs in an integrin-dependent manner ⁷⁴ . It is likely that changes in spatiotemporal interaction between Rab GTPases and specific guanine nucleotide exchange factors contribute to diversify the role of Rab GTPases in axon growth and regeneration.

When the transcriptional landscape of mouse DRG neurons was explored in both growth-competent and -incompetent states at different developmental stages, Cacna2d2, the gene encoding the α2δ2 subunit of voltage-gated calcium channels ⁷⁵ , was identified as a developmental switch that limits axon growth and regeneration ²⁷ ( Figure 2). Interestingly, in these neurons, the developmental transition from a growth-competent (electrically dormant) to a transmitting (electrically active) phase is associated with a marked increase in the expression of genes that control synapse formation and synaptic transmission. Deletion or silencing of Cacna2d2 in adult DRG neurons promotes axon growth in vitro. Pharmacological blockade of α2δ2 via systemic injection of gabapentinoids promotes regeneration of sensory axons after SCI in adult mice ²⁷ . Precisely how gabapentinoids enhance axon regeneration is unknown, but a mechanistic understanding is important, especially since these drugs are often used in humans to treat various neurological disorders, including neuropathic pain. Moreover, a multi-center cohort study found that motor recovery is improved in SCI individuals receiving gabapentinoids ⁷⁶ . Together, these data highlight the need to consider repurposing gabapentinoids as a novel treatment for CNS repair.

During development, a discrete number of transcription factors act as master regulators of gene expression. Among others, Sox11 is highly expressed in many developing organs and its expression is turned off in adults. Whereas Sox11 expression is not changed after CNS injury, its expression increases after peripheral injury, facilitating regeneration of injured peripheral nerves ^{34, 77} . In normally non-regenerative cortical motor neurons, forcing Sox11 expression enables sprouting and regeneration of corticospinal tract (CST) axons after unilateral pyramidotomy and cervical SCI, respectively ²⁸ . However, forced expression of Sox11 in CST neurons impairs, rather than improves, skilled forelimb functions ²⁸ . Thus, improved axon regeneration does not necessarily predict that functional recovery also will improve. Intuitively, this makes sense since functionally significant axon regeneration is a multi-step repair process in which regenerating axons must re-establish proper synaptic connectivity in order to effectively integrate into existing or regrowing neuronal circuitry. Another study tested whether overexpression of Sox11 or other master regulators of gene transcription can enhance regeneration of retinal ganglion cell (RGC) axons after optic nerve crush injury in adult mice. Only overexpression of Sox11, among the seven candidates tested, robustly increased RGC regeneration ⁶⁴ . Interestingly, a gene ontology analysis of transcriptomics data derived from RGC neurons overexpressing Sox11 revealed that most genes that are suppressed by Sox11 are associated with synaptic transmission ⁶⁴ , highlighting similarities with the α2δ2 findings described above. Together, these data suggest that genetic gain-of-function manipulations can rejuvenate adult neurons, enhancing their growth potential; however, these same manipulations may inadvertently impair synaptic function in the neural circuitry.

In a search for mechanisms underlying neural plasticity, a recent study profiled the transcriptome of sprouting intact neurons isolated from mice lacking Nogo receptor 1 (for example, a receptor for myelin-associated axon growth inhibitors) after incomplete SCI. In these mice, structural plasticity and regeneration of CST axons are enhanced in various CNS injury models ^{78, 79} . The authors found that the lysophosphatidic acid (LPA) signaling modulators, LPPR1 and LPAR1, are intrinsic regulators of axon growth in corticospinal neurons. More specifically, LPPR1 overexpression or LPAR1 inhibition promoted collateral sprouting of intact CST axons and enhanced functional recovery after unilateral pyramitomy in adult wild-type mice. LPA is a bioactive lipid species derived from membrane phospholipids, and among the many cellular mechanisms that LPA signaling is known to affect (including oligodendrocyte maturation, myelination, astrocyte proliferation, and inflammation), synaptic transmission is also affected by LPA ⁸⁰ .

Single-cell RNA sequencing has emerged as a powerful technology that enables researchers to identify expression changes of thousands of genes in heterogeneous cell populations ^{81, 82} . A recent study applied this technology to reveal DRG neuron heterogeneity and molecular dynamics after sciatic nerve transection ⁴⁰ . DRG neurons can be classified into several functionally distinct subtypes with very different gene expression patterns ^{83– 85} . Such heretogeneity is reflected by the fact that injury to the peripheral branch of DRG neurons is often associated with mixed responses such as pain, cell death, plasticity, and axon regeneration. After segregating DRG neurons into different subtypes, weighted gene co-expression network analysis revealed injury-responsive gene modules with distinct expression patterns among the different subtypes. Interestingly, the cell death genes—programmed cell death-2 and neuron survival-like ISL LIM homeobox—were upregulated and downregulated, respectively, in a subset of non-peptidergic nociceptor neurons 3 days after injury ⁴⁰ . The fact that caspase-3 was upregulated in all injured subtypes suggests that these neurons may be more susceptible to cell death and therefore not able to regenerate. Indeed, a prerequisite for axon regeneration is that injured neurons survive. Dynamic changes in gene transcription in DRG neuronal subtypes were identified by completing an analysis of the transcriptome at 3 and 7 days after axotomy. Genes related to nervous system development, axonogenesis, regulation of metabolic process, and actin cytoskeleton reorganization were gradually upregulated in large myelinated neurons ⁴⁰ . In contrast, many genes related to learning or memory or nucleus organization were downregulated in these neurons ⁴⁰ , further implicating gene inactivation as an important regulator of axon growth programs (see above).

A phenotypic screen of kinase inhibitors (that is, kinomics) combined with machine learning identified the ribosomal S6 kinase 1 (S6K1) as a negative regulator of axon regeneration in rodents ⁴² . In C. elegans, ribosomal S6 kinase loss of function elicits new axon growth cone formation after injury and accelerates axon elongation ⁸⁶ . In vitro, S6K1 inhibition enhances growth of primary mouse hippocampal neurons. In vivo administration of a selective S6K1 inhibitor (for example, PF-4708671) promotes regeneration of CST axons into and beyond the lesion site in a model of cervical SCI. Functional recovery also is achieved in SCI animals treated with PF-4708671. The benefits of inhibiting S6K1, a known effector of the mammalian target of rapamycin (mTOR), conflict with data showing that mTOR is a positive regulator of axon regeneration in mammals ^{16, 87– 89} . Hence, new kinomics data have enriched our understanding of molecular mechanisms of axon regeneration by showing that PI3K/mTOR signaling is negatively regulated by S6K1.

Growth cones are specialized structures that are required during axon growth and regeneration ⁹⁰ . A better understanding of the signaling pathways that control growth cone activity may be necessary to gain control of axon growth, guidance, and regeneration as well as formation of neural circuits. Reversible protein phosphorylation is one of the most studied post-translational modifications. Phosphorylation of proline, serine, threonine, and tyrosine residues plays a crucial role in function, subcellular localization, and degradation of proteins, thus participating in various cellular processes, including signal transduction. A phosphoproteomic study of growth cone membranes isolated from postnatal day 1 rat forebrain identified 4596 phosphorylation sites from 1223 phosphoproteins ⁴¹ . Of these phosphorylation sites, proline phosphorylation was the most represented. Analysis of the identified phosphoproteins suggested that cytoskeletal components and signaling proteins were the most abundant ⁴¹ . Using a kinase-specific phosphorylation site prediction tool, the authors of this study revealed that proline phosphorylation was due to activation of the mitogen-activated protein kinase (MAPK) pathway ⁴¹ . Of note, coordinated activation of highly conserved MAPK pathways is required for axon growth and regeneration ^{68, 91– 93} . Strikingly, the most abundant phosphorylation site was an uncharacterized serine 96 of the growth-associated protein 43 (GAP-43), which is highly expressed during development and regeneration ^{94– 97} . Of the different kinases involved in signal transduction, c-Jun N-terminus kinase (JNK) was responsible for numerous phosphorylated sites in the phosphoproteomic data set ⁴¹ .

Another interesting study applied quantitative phosphoproteomics to study changes in protein phosphorylation in primary cerebellar granule neurons plated on growth-inhibitory chondroitin sulfate proteoglycans (CSPGs). Of the differentially phosphorylated proteins, phosphorylation increased on 41 peptides and decreased on 77 in neurons exposed to CSPGs ⁹⁸ . Cytoskeletal proteins were the top annotated category, representing 25 of the 118 phosphopeptides identified. Among these cytoskeletal proteins, 14 were of the actin family of cytoskeleton proteins. Cofilin, an actin depolymerization factor regulated by phosphorylation, plays an important role in growth cone behavior and neurite outgrowth ⁹⁹ . In neurons exposed to myelin-associated inhibitor, phosphorylation and inactivation of cofilin have been shown to be regulated via LIM kinase and slingshot phosphatase ¹⁰⁰ , contributing to axon growth inhibition and regeneration failure. Similarly, overexpression of the transcription factor serum response factor enhances axon regeneration through cytoplasmic localization and cofilin-mediated reactivation of actin dynamics in growth-inert retraction bulbs ¹⁰¹ . The top three signaling pathways representing the 118 phosphopeptides were pyrimidine metabolism, p38MAPK pathway, and synaptic vesicle trafficking. Together, these results suggest that phosphoproteomics can serve as a powerful approach to unmask promising targets and signaling pathways to overcome regeneration failure in the adult CNS.

Successful axon regeneration may require a detailed understanding of genetic, proteomic, metabolic, and immunologic functions that occur in the body outside the nervous system. Metagenomics is a collection of high-throughput genetic analyses of transorganismal behaviors and the biosphere. Currently, most metagenomics studies focus on non-eukaryotic microbes, especially those found in the gastrointestinal tract (that is, the “gut”), to learn how these microbes affect organs and cells throughout the body, in both health and disease. Compelling data indicate that microbial metabolites, derived from gut microbes, directly affect the function of neurons and glia in the CNS. How or whether these metabolites will affect regeneration in the injured CNS has not been explored; however, robust and lasting changes in gut microbial communities do occur after a brain injury or SCI ^{102– 105} . Injury-induced change in microbial populations is a potentially novel target for regulating the structure and function of injured neurons. Indeed, the magnitude and diversity of the microbial “payload” are remarkable—unique microbial genes outnumber mammalian genes by about 150 to 1 ¹⁰⁶ , and an impressive number of microbial enzymes and metabolites are already known to affect the metabolism and function of mammalian cells, including those in the immune and nervous systems ^{107– 109} . Thus, it is easy to speculate that metagenomic and metabolomic techniques, when applied in the context of axon regeneration models, will reveal novel roles for microbes in affecting axon regeneration.

Although recent progress in the field of axon regeneration clearly illustrates the power of using omics-based approaches to reveal novel molecular mechanisms to target for therapeutic enhancement of axon growth, we believe that a better understanding of the mechanisms controlling presynaptic biogenesis, synaptic alignment, and connectivity will be necessary to rebuild injured neural circuits in a functionally meaningful way.