The Intrinsic Role of Epigenetics in Axonal Regeneration

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Daniela Palacios*, Maria Teresa Viscomi† Epigenetics and Signal Transduction Laboratory, IRCCS Fondazione Santa Lucia, Rome, Italy* Fondazione Policlinico Universitario A. Gemelli IRCCS, Universita` Cattolica del Sacro Cuore, Rome, Italy†

CHAPTER OUTLINE 14.1 Introduction .............................................................................................................................. 333 14.2 Experimental Models of Axonal Injury in Mammals ...................................................................... 335 14.2.1 CNS ................................................................................................................... 335 14.2.2 PNS ................................................................................................................... 335 14.3 Axonal Injury: Cellular and Molecular Events After Nerve Injury ................................................... 336 14.4 Successful PNS Regeneration and Failure of CNS Regeneration: Cell-Intrinsic and Cell-Extrinsic Factors ..................................................................................................................................... 337 14.4.1 Cell-Intrinsic Factors ............................................................................................ 337 14.4.2 Cell-Extrinsic Factors ........................................................................................... 338 14.5 The Epigenetics of Axonal Regeneration ..................................................................................... 339 14.5.1 DNA Methylation and Hydroxymethylation .............................................................. 340 14.5.2 Histone Modifications .......................................................................................... 341 14.5.3 Noncoding RNAs ................................................................................................. 343 14.6 Therapeutic Interventions Targeting the Epigenome to Promote Axonal Regeneration .................... 346 14.7 Conclusions and Perspectives .................................................................................................... 347 List of Abbreviations .......................................................................................................................... 348 References ........................................................................................................................................ 348 Further Reading ................................................................................................................................. 354

14.1 INTRODUCTION During development, neurons of the central nervous system (CNS) and of peripheral nervous system (PNS) extend axons and establish connections with their targets that are often located far away. As CNS neurons mature, growth programs controlling axon extension go dormant and are incompletely Epigenetics and Regeneration. https://doi.org/10.1016/B978-0-12-814879-2.00015-7 # 2019 Elsevier Inc. All rights reserved.

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activated following injury. This decline in not observable in PNS axons1, 2 and this make the PNS more permissive to axonal regeneration after injury. Consequently, failure of damaged adult CNS axons to regrow results in permanent disabilities for individuals with traumatic brain and spinal cord injury or stroke. For several decades, a great effort has been made to disentangle the players and the mechanisms underlying the differences in regenerative competence between PNS and CNS. The initial findings have driven researchers to focus on the extrinsic factors with an inhibitory effect on axon regeneration3 and many molecules have been identified. With the identification of several growth-inhibitory factors associated with CNS myelin and with the glial scar, such as myelinassociated glycoprotein (MAG), Nogo proteins, oligodendrocyte myelin glycoprotein, semaphorins and ephrins, and chondroitin sulphate proteoglycans (CSPGs)3, 4 the regeneration failure has been mainly attributable to environmental inhibitory factors. The discovery that these inhibitory molecules are found to a lesser extent in the mammalian PNS,5 where regeneration occurs, has further supported this hypothesis. However, the lack of axonal growth in adult mammalian CNS injured neurons, even when provided with a growth-permissive substrate,4, 6 and the limited success obtained by strategies based on neutralizing inhibitory factors of the CNS environment,7 has moved the attention to cell-intrinsic factors involved in modulation of the regenerative response. In further support of this, recent in vivo 2-photon imaging studies of laser-mediated axotomy in mouse cerebral cortex showed the inability of mature cortical neurons to regenerate even in a glial scar-free environment.8 Furthermore, more recently, Andersen and colleagues9 showed that the glial scar rather than being hostile to axon growth can promote regeneration in the rodent spinal cord. Together these studies support the idea that the lack of regeneration in the adult CNS is an endogenous property of the injured mature neurons. Therefore a major challenge has been to define the underlying cellular and molecular mechanisms that determine neuronal intrinsic regenerative failure, with the aim of developing therapeutic strategies for removing axons’ roadblocks in the adult mammalian CNS. One of the main intrinsic barriers to functional regeneration of mature mammalian neurons is the epigenome. Whereas in the PNS, injury unlocks mature neurons’ intrinsic axonal growth transcriptional program, this does not occur in the CNS.10 Epigenetic regulation plays a pivotal role in various CNS physiological and pathological processes by regulating gene expression, such as development, apoptosis, proliferation, differentiation, and regeneration.11 In particular, in the field of axonal regeneration, given the importance of chromatin structure for the accessibility of transcription factors and in the transcription of genes needed for axonal regeneration, epigenetic mechanisms are emerging as a powerful approach to removing intrinsic brakes limiting CNS regeneration and for reprogramming mature neurons to an axon regeneration-competent state. Although epigenetic studies in the field of axonal regeneration are quite recent, the growing body of evidence is very promising and highlights the potential key role for epigenetic regulation in axonal outgrowth and regeneration in the adult CNS. These findings are quite interesting and may have broad implications for regenerative medicine in a variety of neurological disorders, including traumatic brain injury, spinal cord injury, and stroke.12 Here we will first introduce a few commonly used CNS and PNS experimental models of axonal injury and summarize the retrograde signaling triggered by axonal injury, and then summarize the latest findings implicating epigenetic mechanisms in axon PNS and CNS regeneration and discuss the therapeutic potential of targeting epigenetic regulators to improve neural recovery.

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14.2 EXPERIMENTAL MODELS OF AXONAL INJURY IN MAMMALS 14.2.1 CNS Among rodent-based CNS axonal injuries, optic nerve (ON) injury and spinal cord injury (SCI) have been mostly employed to study axon regeneration. However, for axon regeneration studies, partial lesion rather than complete lesion of entire axon tracts is more suitable. This choice is dictated by the fact that in the complete lesion paradigm the large number of axons simultaneously lesioned makes it difficult to dissect the injury responses at the level of each axon.

14.2.1.1 Optic nerve lesion model A widely used model of CNS injury is the transection of the ON, representing a unique and highly reproducible model for the study of axonal response. Because of its structural simplicity and fast surgical accessibility, the system is widely used as a model for the study of axonal degeneration/regeneration and for remote death.13 Moreover, the ON has clinical relevance for the pathogenesis of optic neuritis, glaucoma, Leber’s optic atrophy, and trauma.14 In adults, ON injury results in delayed death and apoptosis of retinal ganglion cells (RGCs), the only projecting neurons of the retina, whose axons form the optic nerve and transmit visual information to the brain, causing visual loss.13 After ON injury, RGCs are normally unable to regenerate their axons and die, subsequently inducing the functional loss of vision.15–17

14.2.1.2 Spinal cord (hemisection) injury model Another model of CNS injury widely used is partial or complete spinal cord transection, paradigms particularly useful to study supraspinal responses to injury, axonal regeneration, and subsequent functional recovery.18, 19 Although complete spinal cord transections are relatively easy to perform, and if properly done, interrupt all ascending and descending pathways so there is no issue of spared axons, spinal cord hemisection is generally adopted. Spinal cord hemisection is simple and is associated with low mortality and simple postoperative care. It is not debilitating, because it does not cause the bladder, bowel, or respiratory dysfunction that is generally observed after complete cord injury. It typically damages the cervical or thoracic cord. Lesions at the cervical level involve the upper extremities, causing severe impairments, whereas thoracic-level injuries impede only lower limb function.19 A spinal cord hemisection approach allows the study of remote responses to injury20 and the mechanisms that govern the inhibition or successful regeneration of axons as well as of the resulting functional deficits and potential recovery.19 Interrupted descending and ascending axonal tracts have debilitating consequences, and although proximal segments typically survive, they do not regenerate spontaneously.19 Regenerative strategies known thus far, as well as identified intracellular pathways and growthinhibiting factors, are largely similar to those characterized in ON regeneration.

14.2.2 PNS 14.2.2.1 Dorsal root ganglia injury model PNS studies on axon regeneration have focused on the axons from sensory neurons in dorsal root ganglia (DRG) and in sympathetic ganglia. Sensory neurons in the DRG are particularly interesting for comparing the differential regenerative responses of the PNS versus the CNS and provide a

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favorable model for studying mammalian axon regeneration. This approach is simple and has a high degree of reproducibility. Each DRG neuron extends a unipolar axon that splits into two branches: one peripheral innervating targets such as skin and muscles and a central relaying the sensory information to the CNS, via the spinal cord. While injured peripheral axons are able to regenerate, the central branch fails to do so.21 However, if axotomy of the peripheral branch occurs prior to central branch axotomy (a preconditioning lesion), regeneration of the central axons is greatly enhanced.22, 23

14.3 AXONAL INJURY: CELLULAR AND MOLECULAR EVENTS AFTER NERVE INJURY In both physiological and physiopathological conditions, the body and the axon of neuronal cells communicate.24 In particular, after axonal damage, axon-cell body communication contributes to retrograde responses to injury.20, 25 Axonal injury produces effects at the site of injury and also at the remote level, in particular in the nucleus. Axonal damage triggers molecular signals that travel from the site of damage back to the neuronal cell body to activate specific cellular programs to coordinate local reorganization and communication with the cell body.26 In turn, the results of the new transcriptional program must be communicated back to axons to execute changes for axon maintenance/degeneration.25 These events, collectively termed “cell body responses,” are elicited by the axonal injury and are critical for both axon and neuron fate.26 Notably, although many signaling are activated after axonal injury, differing significantly between CNS and PNS some aspects are common and are crucial for the final response to injury. Injury leads immediately to a rise in intracellular calcium concentration27 at the primary site of injury, where increased calcium levels are fundamental to regulate cytoskeletal disassembly and axonal degeneration upon injury.28 Then the local increase in calcium concentration propagates along the axon and alerts the soma of the injury. Once the soma has been alerted of the downstream damage, the nucleus has a crucial role in priming retrograde injury signaling.24 Changes in intracellular calcium levels activate downstream effectors that in turn regulate cell life-and-death decisions.23, 27 Several injury-responsive signaling proteins, including extracellular-signal-related kinases (ERKs), DLK, JUN N-terminal kinase (JNK), and the transcription factor signal transducer and activator of transcription 3 (STAT3), are retrogradely transported.24 In the PNS these alterations in transcription factor activity result in changes of gene expression in the injured neuron, with modulation of a growing list of “regeneration-associated genes” (RAGs) that is lacking following CNS injury (for a review see Ref. 29). Furthermore, recent data suggest that an early calcium wave elicits epigenetic changes in the soma of injured neurons30 as we will discuss further in Section 14.5 of this chapter. In summary, axon injury leads to the activation of several signaling pathways and, in many cases, of transcription factors or epigenetic changes that convey specific postinjury cellular changes from the site of axon injury to the soma that regulate cell life-and-death decisions. As multiple responses occur after axonal injury, a major challenge is to understand how the generation and propagation of these injury signals are received and integrated in cell bodies, in order to develop specific therapeutic protocols for promoting axonal regrowth after injury.

14.4 CELL-INTRINSIC AND CELL-EXTRINSIC FACTORS

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14.4 SUCCESSFUL PNS REGENERATION AND FAILURE OF CNS REGENERATION: CELL-INTRINSIC AND CELL-EXTRINSIC FACTORS During nervous system development, embryonic neurons are equipped with a high axon growth potential, but with maturation, some adult neurons retain the ability to self-repair and are capable of reverting to a growth state through a transcription-dependent process31 while some other neurons lose their capacity to mount a regenerative response. The developmental decline of axon growth ability leads to a key difference between PNS and CNS neurons. PNS neurons are considered regeneration-competent because they retain the regenerative ability that is reactivated by injury. Conversely, CNS neurons are considered regeneration-incompetent neurons because they lose their capacity to mount a regenerative response. This difference was known and intensively studied more than 100 years ago by Ramo´n y Cajal, and although many hypotheses have since been postulated to explain the different response of PNS and CNS neurons to injury, we are still far from unraveling the exact programs required for axon regeneration. Differences in the availability or recruitment of one or multiple intrinsic regeneration-promoting mechanisms, including priming by calcium waves, epigenetic modifications, local mRNA translation, retrograde transport of transcription factors, or the presence of extrinsic mechanisms, including the surrounding environment, have been described to explain the limited response of CNS neurons to injury,32 giving rise to two opposite currents: supporters of either cell-intrinsic factors or cell-extrinsic factors. Nowadays, although we are still far from elucidating the factors required for axon regeneration, the body of knowledge gathered supports the view that in CNS neurons, the combined action of extrinsic factors limiting axonal growth, the limited injury-signaling mechanisms, and the lack of robust expression of regeneration-associated genes (RAGs) together hinder axon regrowth after injury. Conversely, the regenerative capacity of axons in the PNS is supported by the combination of cell-extrinsic and cell-intrinsic factors that generate a permissive milieu for a successful axonal regrowth.

14.4.1 Cell-Intrinsic Factors The intrinsic cellular pathways that are activated after injury differ between PNS and CNS neurons and evidence from multiple studies supports the point of view that the weak intrinsic regenerative ability of mature neurons is the major impediment to axon regeneration in the CNS. In the PNS, the regenerative response is dependent on transcriptional events occurring in the neuronal cell body following an injury event occurring in the axon.33 Specialized signaling mechanisms are required for transmitting information from the lesion site to the cell soma. Studies on peripheral sensory neurons have provided compelling evidence for the importance of retrogradely transported injury signals for initiation of a regeneration response.26, 34 These retrograde injury signals have to travel from the injury site back to the cell body to elicit transcriptional changes and to generate a multifaceted response to injury. After growth cone formation, injured axons start to regenerate or sprout. Earlier studies indicated that axonal regeneration or sprouting is accompanied by reactivation of genes that are normally expressed during development.29 In particular, axotomy of PNS neuron induces broad and coordinated

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gene transcription, namely regeneration-associated genes (RAGs) that is lacking following CNS injury (for a review see Refs. 29, 35). DRG neurons have provided an important model for demonstrating how RAGs induction allows axon regeneration. Axonal injury of the peripheral projecting branch induces the expression of RAGs, whereas injury to the central projecting branch does not. Interestingly, in this model a peripheral conditioning lesion allows regeneration of a subsequently injured central branch23 and this last effect has been associated with transcription of RAGs induced by peripheral axon injury that permits regeneration to an otherwise regeneration-lacking axon.36 Together, these observations indicate that manipulations increasing expression of RAGs in the CNS can also promote regrowth of “resistant” axons into transplanted peripheral nerve segments.37, 38 However, not all types of CNS neurons exhibit this capacity, and those that regenerate upregulate expression of RAGs in the presence of the graft.38 In recent years many candidate RAGs have been identified by using genomic or proteomic approaches.39–41 Individually identified RAGs span many functional categories of genes, ranging from genes that encode adhesion/guidance molecules (i.e., integrin) to structural and cytoskeletal-associated proteins (i.e., GAP43).29 The contribution of specific RAGs to axon regeneration/growth (either in PNS or CNS neurons) has been assessed both in vitro and in vivo by evaluating their role in the regenerative response. These studies often show that knockdown of individual downstream RAGs results in small decreases, or delays, in peripheral regeneration, suggesting that the modulation of any single downstream RAG may be insufficient to achieve effective, long-distance axonal regeneration.42 Considering that several genome-wide studies have identified numerous genes that are upregulated or downregulated following a neural injury,41, 43 it is conceivable that only the manipulation of multiple signaling pathways can activate robust axonal outgrowth and regeneration.44, 45 Several pieces of evidence demonstrate that the difference in the regulation of transcription between the PNS and CNS is not limited to a subset of RAGs. Although a reduction in the regenerative ability of CNS neurons is mainly due to changes in the transcriptional program, reduction in chromatin accessibility at gene regulatory regions represents an intrinsic obstacle limiting the regenerative capacity of neurons.30, 46, 47 To test the emerging link between chromatin structure and axon growth, previous studies have genetically or pharmacologically manipulated chromatin accessibility in functional assays of axon growth. Interestingly, several findings indicate that chromatin structure at genes involved in regenerative axon growth undergoes profound restriction as neurons mature35 and that genes that are not actively transcribed, such as RAGs in nonregenerating CNS neurons, may be held in a “closed” chromatin state, rendering them inaccessible to transcription factors.46, 47 This last evidence suggests that the regulatory mechanisms triggering epigenetic regenerative responses are not functional in the CNS, thus the gene expression changes required for axonal regeneration are blocked.35 Considering the great interest in several therapeutic approaches in unlocking CNS axon regeneration, and given the difficulty in directly upregulating gene expression in vivo, the action of epigenetic modulators has gained significant interest in regenerative medicine. This aspect will be considered in Section 14.5.

14.4.2 Cell-Extrinsic Factors In addition to the intrinsic insufficiency of mature neurons to regrow axons, one of the major differences between the CNS and PNS is the surrounding environment in which the injured axons try to regenerate. These extrinsic inhibitory molecules are not present within the PNS, where axon regeneration robustly occurs after injury. As with the CNS, many factors derived from various supporting cells,

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including oligodendrocytes and astrocytes, contribute to the creation of a growth-inhibitory environment after injury either by forming physical barriers or, alternatively, by receptor-mediated repulsion axons.4, 48, 49 In particular, in the CNS a glial scar is formed upon injury by migration of astrocytes, proliferation of reactive astrocytes, and accumulation of intermediate filament proteins such as the glial fibrillary acidic protein (GFAP), vimentin, and others.50 The glial scar at the site of injury represents another insurmountable obstacle to axon growth, and this barrier is the result of production of chondroitin- and keratin-sulfate proteoglycans (CSPGs, KSPGs) (for a review see Ref. 50). However, several studies demonstrate that counteracting or removing the external inhibitory molecules is insufficient for long-distance axon regeneration, as demonstrated in studies on functional interference with CSPGs or myelin-based inhibitors.51 All of the aforementioned processes are regarded as extrinsic cues that prevent CNS regeneration. However, although the dogma on cell-extrinsic factors limiting CNS regeneration has been accepted for several decades, a recent study shows that glial scars, particularly newly generated immature scar-forming astrocytes, rather than being hostile to axon growth can promote regeneration in the rodent spinal cord.9 Interestingly, Anderson and colleagues have shown that sustained delivery of required axon-specific growth factors that is not adequately expressed in spinal cord lesions, combined with the activation of neuron-intrinsic growth programs, can promote robust regrowth of injured axons, despite the presence of inhibitory cues, and that this stimulated axon regrowth can occur either in direct contact with astrocyte scars or independently of astrocyte processes.9 Together, these findings have questioned the prevailing dogma that an astrocytic scar is the principal cause for the regrowth failure of injured mature CNS axons and have important implications for CNS repair strategies. The findings may also represent exploitable bridges for regrowing axons after CNS injuries, and furthermore clearly demonstrate that axon regrowth after injury requires important and coordinated intrinsic and extrinsic events, with a balance of extrinsic and intrinsic regulatory cues that modulate one another’s effects on axon regeneration.

14.5 THE EPIGENETICS OF AXONAL REGENERATION As discussed above, one of the main determinants of axonal regeneration is the ability of the PNS, but not the CNS, to robustly modulate gene expression in response to injury. Being at the interface between the genome and the environment, the epigenome stems as one of the main intrinsic regulators of the regeneration-associated transcriptional program.12, 52 Amongst the components of this program, RAGs play a fundamental role in initiating and establishing the regenerative response. The impaired regenerative ability observed with age correlates with overall chromatin changes that restrict the activation of RAGs, providing a further causal link between the epigenome and the degree of regenerative response.53 Dissecting the epigenetic and chromatin landscape that favors regeneration is therefore of major interest and has been the subject of an increasing number of studies in the past few years. A summary of such studies is discussed here, highlighting the potential applications of epigenetic interventions aimed at increasing axonal regeneration not only in the PNS but also in the regeneration-resistant CNS. Compared to other tissues, the epigenetic response elicited upon axonal injury has been far less characterized, likely due to a lack of adequate cell lines for the different types of neurons and the difficulty in obtaining enough purified cells for unbiased epigenetic analysis. In fact, most studies so far have used whole tissue for analysis. In those experiments, contamination of neuronal cells with populations of nonneuronal cells, such as the abundant glia compartment, impose caution when interpreting the data.

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14.5.1 DNA Methylation and Hydroxymethylation Five-methylcytosine (5meC) within the context of CpG dinucleotides is the most abundant and one of the most studied epigenetic modification in mammals.54 It is mainly a repressive modification that restricts gene expression through either the recruitment of methyl binding proteins and corepressor proteins, or by interfering with the binding of some transcription.55–58 Details on the molecular mechanisms and players that regulate DNA methylation are provided elsewhere in this book. Here we will discuss current knowledge regarding the contribution of the DNA methylation machinery to axon function and regeneration. Expression of several DNA methyltransferases (DNMTs) is high in neural cells and changes in the DNA methylome have been associated to neural plasticity in a variety of conditions, ranging from development and neuroprotection to neuropathic pain and cognitive and behavioral functions.59–62 However, its role in axonal regeneration has been less explored. Recently, a temporal DNA methylome analysis was performed in DRG cells after either peripheral (sciatic nerve) or central (spinal nerve) axotomy using methylated DNA immunoprecipitation (MeDIP) followed by array hybridization.47, 63 Bioinformatic analysis identified a small number of differentially methylated regions (DMRs) amongst regenerating and nonregenerating lesions. Surprisingly, none of the commonly investigated RAGs was associated with changes in the methylome in these experiments. However, caution is needed when interpreting these results, as analysis was done on whole DRG, leading to abundant glial contamination.47, 63 In a different study, interfering with the DNA methylome through treatment of mice with folic acid, an activator of the synthesis of the methyl group donor, S-adenosine methionine (SAM), stimulates CNS regeneration after combined spinal cord and sciatic nerve lesions in rats. Folate treatment shows a dose-dependent, biphasic effect that correlates with gene-specific changes in the DNA methylation profile.64 Although traditionally it was considered a very stable covalent modification, extensive work from the past two decades has shown that 5meC can be dynamically removed through the action of the Tet-eleven translocation (TET) methylcytosine dioxygenases.65, 66 TET enzymes oxidize 5meC to 5-hydroxymethylcitone (5hmC). 5hmC acts as an epigenetic mark itself or is further oxidized into 5-formyl-cytosine (5fC) and 5-carboxycytosine (5caC), followed by the thymidine DNA glycosylase (TDG)-mediated base excision pathway, leading to active DNA demethylation.67 5hmC is abundant in the CNS when compared to other tissues and, as also occurs for DNA methylation, it plays fundamental roles in neurodevelopment, cognitive and behavioral functions, and several neurological disorders.68–71 Despite its abundance in neural tissue, its contribution to axonal regeneration has begun to be addressed only recently. Initial work from Ming’s lab suggested 5hmC is necessary for cellular reprogramming upon nerve injury. Their work showed that upon PNS lesion there is an increase in TET3 levels and global 5-hmC in DRG neurons.10 Amongst the genes targeted by these modifications, the authors identified several RAGs, the expression of which is increased and contributes to the regenerative response. Consequently, both TET3 and TDG are necessary for successful regeneration. Moreover, the increased TET3 levels and associated 5hmC in DRG depends on the retrograde calcium signal, suggesting that injury-mediated calcium intake regulates DNA demethylation.10 By using a genome-wide approach consisting of immunocapture of 5hmC followed by nextgeneration sequencing analysis (NGS) Loh et al. mapped the 5hmC profile in regenerating versus nonregenerating branches of the DRG. As expected, the two insults triggered different 5hmC changes,

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which correlated with the modulation of specific signaling pathways known to regulate axonal growth, such as the phosphatase and tensin homologue (PTEN) and the bone morphogenetic protein (BMP) signaling pathways.72 Interestingly, an increase in 5-hmC in brain with age was described by Szulwach and collaborators, which affects mainly genes dynamically modulated during development and aging.73 If this increase in basal levels of 5hmC correlates with the impaired regeneration ability that occurs with age needs to be further investigated.

14.5.2 Histone Modifications DNA inside a eukaryotic nucleus is wrapped around histone proteins into a highly compacted structure called chromatin. On the one hand, such compaction is needed to fit the 2m-long DNA molecule into the limited space of the nucleus. On the other hand, this implies that for nuclear processes (including transcription) to occur, this structure needs to be partially disrupted.74 Posttranscriptional modification of histones acts by modulating the chromatin accessibility and represents the second level of modulation of the epigenome. Chapter 3 of this book describes in detail the different posttranscriptional modification of histones, the molecular players involved and describes how the combinations of different histone modifications (the so-called histone code) finely regulate the transcriptional output of a cell. Here, we will focus on the role of histone modifications, and in particular histone acetylation and methylation, in the regulation of axonal regeneration and we will highlight the differences between regenerating (PNS) and nonregenerating (CNS) neurons. Initial work using immunofluorescence and biochemical analysis showed a global increase in histone acetylation upon peripheral axotomy46 but not upon central axotomy.75 Interestingly, when using DRG neurons as a model, it was observed that only damage to the peripheral branch, but not to the central branch, elicits a hyperacetylation response in the cell nucleus. However, if a preconditioning lesion is performed, an increase in acetylation can be also observed upon axotomy of the central branch.46 A more detailed characterization of such a response by chromatin immunoprecipitation (ChIP) analysis at discrete loci showed an increase in H4 acetylation at the regulatory regions of several RAGs, which correlated with gene activation.46 Despite the fact that the CNS is known for its inability to mount a regenerative response upon nerve injury, it has been shown that depletion of key molecules, such as PTEN or the transcription factor Krupper-like factor 4 (KLF4), is enough to induce axon regeneration in RGCs upon ON injury.76, 77 This indicates that the intrinsic inability of the CNS to regenerate can be modulated by specific interventions. In this line, work from di Giovanni’s lab showed that treatment with the pan class I/II HDAC inhibitor (HDACi) trichostatin A (TSA) induces reactivation of several HATs such as CREB binding protein (CBP), p300, and p300/CBP-associated factor (PCAF) in cultured neurons and this mediates neurite outgrowth.78 However, different to what observed in vitro, TSA treatment induces CBP expression and p53 acetylation in an in vivo model of ON injury, but does not modulate the expression of p300 or PCAF.75 Despite reactivation of CBP and increased survival of RGCs, TSA was not enough to promote axon growth in this model of CNS injury, suggesting different epigenetic modifiers modulate cellular functions such as axon regrowth or cell death in the CNS.75 Consistent with this idea,

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overexpression of p300 alone is enough to induce axonal growth in a mouse model of ON injury, through a mechanism involving H3, p53, and CCAAT enhancer-binding protein (C/EBP) acetylation.75 p300 overexpression, on the contrary, does not induce survival of RGCs.75 More recent work from the same lab revealed that PCAF is upregulated after a peripheral, but not central, lesion, where it promotes axon growth through hyperacetylation of a subset of RAGs. Furthermore, they demonstrated that the nuclear function of PCAF in axotomized neurons is regulated by a retrograde signaling driven by the extracellular signal-regulated kinase (ERK), which is activated upon nerve lesion.47 These observations are in agreement with the idea that the epigenome acts as a sensor of the regenerative environment through the action of intracellular signaling cascades.79 Finally, PCAF overexpression is enough to induce a regenerative response also in the CNS after SCI.47 Histone acetyltransferases’ (HATs) activity in eukaryotic cells is counterbalanced by the action of three families of histone deacetylase (HDAC) enzymes. Several studies have now demonstrated that class I histone deacetylases are dynamically regulated upon axonal injury and functional studies were fundamental to dissect individual contributions. Combined genetic depletion of Hdac1 and Hdac2 in RGCs contributes to neuroprotection upon ON injury by inhibiting the p53/PUMA proapoptotic pathway,80 while a different study showed Hdac3 depletion and pharmacological targeting in the same system ameliorated the atrophic phenotype and reduced cell death.81, 82 Moreover, HDAC3 has been shown to interact with class II HDAC4 and HDAC5 in the PNS. HDAC3 interaction with HDAC5 is necessary for the nuclear export of the former, a mechanism that contributes to the regenerative response and is altered in the CNS.30 For some of these HDACs, a function outside the neuronal compartment has also been described. For instance, HDAC2 has been shown to regulate the activity of Schwann cells, the cells responsible for myelin production in peripheral axons, upon PNS injury through a mechanism involving chromatin remodeling and the regulation of the key determination factor Oct6. Genetic and pharmacological inhibition of the enzyme accelerates functional recovery and increases regeneration in rodents.83 Another nuclear factor the activity of which has been shown to be fundamental for the function of Schwann cells and regeneration in the PNS is NK-kB.84 Given that NF-kB activity depends on its deacetylation by HDAC1/2, these enzymes may be involved in the temporal regulation of both dedifferentiation and subsequent remyelination phases of Schwann cells.85 Finally, ChIP-seq analysis performed in Schwann cells upon PNS injury revealed a dynamic regulation of active, H3K27Ac-containing enhancers that may be involved in coordinating the regenerative response.86 In addition to class I HDACs, much attention has been put on calcium-dependent histone deacetylases, or class II HDACs, in the nervous system. Class II HDACs are the major calcium-sensor at the epigenome and as such play a fundamental role in several neural functions. From the mechanistic point of view, when calcium levels are high inside the cell, phosphorylation of HDAC4 and 5 by the calcium/calmodulin-dependent protein kinase II (CAMKII) and other kinases induces a nucleus-tocytoplasm switch through a mechanism involving the 14-3-3 chaperone protein.87, 88 Upon axon injury in DRG neurons, a calcium wave originating from the injury site propagates backwards towards the cell soma, causing the nuclear export of the histone deacetylase 5 (HDAC5) in a PKCμ-dependent manner, leading to enhanced histone acetylation.30 Importantly, injury-induced HDAC5 nuclear export enhances histone acetylation to activate a proregenerative gene expression program that fails to occur in RGCs, a model of CNS axonal injury. A third class of HDACs, class III (NAD +-dependent) HDACs or sirtuins are important sensors of the metabolic state of the cells.89 Despite the fact that their function in axonal regeneration has been less

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studied, some studies point to these enzymes as key regulators of axon outgrowth, degeneration and regeneration through both nuclear and cytoplasmic functions.90–94 A recent study showed that all family members of Sirt1–7 increased upon ON injury, but its role in this model has not yet been addressed.95 A role in preventing axonal degeneration has been described for SIRT1, through a mechanism involving the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme NMNAT1.94 In addition, SIRT1 has also been involved in axonogenesis93 and axon regeneration upon sciatic nerve injury.92 Finally, and in agreement with the fundamental role of sirtuins as metabolic sensors, a role in sensing the energy imbalance associated with aging has been unveiled for SIRT2, which leads to axonal degeneration in aged organisms.96 Similar to what is observed for HATs, nonhistone targets of several HDAC family members have been identified. This dual role of HDACs, acting both at nuclear and cytoplasmic targets unveils the complexity of the system and the need for a coordinated response both at the nucleus and the cytoplasm for proper regeneration. Amongst the nonnuclear targets of HDACs several cytoskeletal components have been identified. For instance, deacetylation of alpha-tubulin by HDAC597 or SIRT290 modulates dynamics of microtubules in peripheral neurons. In addition to histone acetylation and deacetylation, recent work has begun to unveil the contribution of Polycomb complexes to axonal regeneration. Polycomb repressive complexes (PRCs) are epigenetic modifiers that act through transcriptional repression. There are two main PRC complexes, PRC1 and PRC2. The canonical Polycomb-mediated gene expression starts with the action of EZH2, the methyltransferase activity of PRC2. In stem and progenitor cells, EZH2 di- and trimethylates lysine 27 in histone 3. H3K27me3 creates a docking site for PRC1 which stabilizes gene repression through several mechanisms including histone 2A (H2A) ubiquitination by the E3 ubiquitin ligase RING1, and chromatin compaction and interplay with other epigenetic modifiers such as the DNA methylation machinery.98 In a recent study by Duan et al., the authors show that Polycomb chromobox (CBX) 2, 7, and 8 proteins, fundamental components of PRC1, act as a brake to axon regeneration of DRG neurons. Furthermore, they identified Gata4 and Sox11 as two genes downstream of CBX7 in the regeneration pathway.99 Despite this, more work needs to be done to further clarify the role of other Polycomb proteins in axonal regeneration, and this study opens up the possibility that interfering with Polycomb-mediated gene silencing may represent a new pharmacological tool for regeneration in the CNS (Fig. 14.1).

14.5.3 Noncoding RNAs Both long and small noncoding RNAs have been shown to regulate axonal regeneration, though the molecular circuitries affected are only starting to be dissected. Here we will discuss some of the noncoding RNA-regulated circuitries involved in PNS axonal regeneration and we will highlight putative candidates for therapeutic interventions.

14.5.3.1 MicroRNAs MicroRNAs are small noncoding RNAs that act as posttranscriptional repressors of gene expression through binding to target mRNA at their 30 untranslated region (UTR).100 Given their key role in the establishment and maintenance of discrete transcriptional programs, miRNAs have been the subject of intensive study in recent years. The canonical biogenesis of miRNAs is a tightly regulated process that initiates in the nucleus with the transcription of the primary miRNA (pri-miRNA) and continues

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CNS • In SCI •

PNS

PCAF over-expression HDACi (VPA, TSA, MS-275)

CENTRAL BRANCH

Folic acid treatment (after combined lesion)

AcH3, AcH4 after pre-conditioning lesion

DMRs in peripheral vs central branch

HDAC5 neclear export (Ca2+- and PKCµ-dependent)

PERIPHERAL BRANCH PCAF, AcH3, AcH4 (ERK-dependent)

In ON

•

p300 over-expression

•

HDACi (TSA treatment)

• •

HDAC1, HDAC2 depletion Genetic/pharmacological inhibition HDAC3

TET3 and 5hmC (Ca2+ -dependent) H3K27Ac at enhancers in Schwann cells • • •

Genetic/pharmacological inhibition HDAC2 (in Schwann cells) Genetic depletion of CB´7 (PRC1) SIRT1 over-expression

FIG. 14.1 Schematic representation of the different epigenetic responses elicited upon CNS and PNS axonal injury and the effects of genetic/pharmacological modulation of the epigenome on axonal regeneration or on neuronal survival. The neuronal cell on the left represents a neuron of the CNS. In blue we represent the experimental manipulations of the epigenome (genetic and/or pharmacological) that induce axonal regeneration. In red are represented the experimental manipulations (genetic and/or pharmacological) that induce neuronal survival. The neuronal cell on the right (PNS) represents a sensory neuron in dorsal root ganglia, in which the two branches are visible: one peripheral, innervating targets such as skin and muscles, and one central, relaying the sensory information to the CNS, via the spinal cord. In black are represented the different epigenetic responses observed after axonal injury. In blue, the different epigenetic interventions (genetic and/or pharmacological) that induce axonal regeneration. Abbreviations: AcH3, acetylated histone H3; AcH4, acetylated histone H4; DMRs, Differentially methylated regions; HDAC, Histone deacetylase; HDACi, HDAC inhibitor; ON, optical nerve injury; PCAF, p300/CBP-associated factor; RGCs, retinal ganglion cells; SCI, spinal cord injury; TET, Tet-eleven translocation; TSA, Trichostatin A; VPA, Valproic acid.

through the sequential cleavage of this long transcript by two RNase III-containing complexes. First, Drosha, a component of the Microprocessor complex, cleaves the primary transcript to generate the precursor miRNA (pre-miRNA) hairpin. This first cleavage occurs cotranscriptionally at the chromatin of target pri-miRNA.101 Then the pre-miRNA is exported to the cytoplasm where Dicer-containing complexes cleave near the loop and generate a miRNA:miRNA* duplex of around 22 nucleotides.

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These duplexes are finally loaded into the Ago2-containing RISC complex where they pair with target mRNAs to either induce their degradation or inhibit translation.102, 103 Interference with the miRNA-processing pathway through conditional deletion of Dicer blocks regeneration upon peripheral nerve damage.104 Dicer is also essential for the function of Schwann cells, as specific depletion of this cell compartment arrests cell differentiation causing severe neurological defects similar to those observed in congenital hypomyelination.105–107 Injury-activated miRNAs in Schwann cells were predicted to target genes involved in differentiation and proliferation processes.106 The role of miRNAs in the peripheral system has been recently reviewed.12, 108 Several miRNAs have been shown to modulate axonal regeneration either by promoting (i.e., mir-26a, mir 30b, mir210, etc) or suppressing it (mir-138, mir-155, let, etc.). A summary of the axonal regenerationassociated miRNAs identified to date is given in Table 14.1. It is likely that this number will increase in the coming years. Finally, alteration of miRNA levels has been associated with impaired homeostasis and regeneration in several tissues, including the nervous system. Similarly, Zou et al. identified a key regulator of the developmental clock in C. elegans consisting of a negative feedback circuitry involving the late onset miRNA let-7 and its target lin-41, which encodes for the tripartite motif (TRIM) protein. Increased let-7 with age repressed lin-41 in old neurons, leading to a progressive decline in regeneration potential.121

14.5.3.2 Long noncoding RNAs (lncRNAs) lncRNAs are particularly important for the mammalian nervous system, being involved in neural development, cognitive and behavioral functions, and neural regeneration.122, 123 Some of them act as epigenetic regulators of gene expression through mechanisms ranging from their role as quenching

Table 14.1 MiRNAs Modulating Axonal Regeneration miRNA

Target mRNA

Location

Functiona

Reference

Mir-138

Sirt1 Vimentin Gsk3β Sema3a RhoA Ngf Not determined Efna3 Pten Bdnf Egfr Klf-7 Kremen

DRG neurons Schwann cells DRG neurons RGC Supraspinal neurons Schwann cells DRG neurons and macrophages DRG neurons Spinal cord Spinal cord ON Schwann cells DRG neurons

Negative Negative Positive Positive Positive Negative Negative Positive Positive Negative Negative Negative Positive

92 109 110 111 112 113 114 115 116 117 118 119 120

Mir-26a Mir-30b Mir-133b Let7 Mir-155 Mir-210 Mir-29a Mir-21 Mir-146b Mir-431

a Indicates their positive or negative role on axonal regeneration and/or proliferation and differentiation of Schwann cells. MiRNA and target mRNAs involved in axonal regeneration.

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molecules for miRNAs (as a sponge) to modulation of the epigenetic machinery.124 Using a microarray platform, Yu et al. identified 105 lncRNAs that are differentially regulated at different stages after axotomy of DRG neurons. Further, through bioinformatics analysis, they mapped a coexpression network of lncRNAs and coding genes. Interestingly, amongst the putative targets of the differentially expressed lncRNAs, they found genes involved in neuropeptide signaling and glia cells’ migrations, suggesting they could be involved in regulating the regenerative response following nerve lesion.125 Despite this, further studies are needed to dissect the contribution of lncRNAs to axonal regeneration and to identify their individual mechanisms of action. This work highlights the molecular complexity at the basis of axon regeneration. Finally, uncovering a further layer of complexity, posttranscriptional modification of RNA (both coding and noncoding) is starting to emerge as a key regulator of axonal regeneration, highlighting the contribution of the epi-transcriptome to the modulation of the regenerative response upon PNS injury. In particular, a pioneer study showed a dramatic increase of N6-methyladenosine (m6A) at mRNAs in the DRG upon sciatic nerve lesion.126 Furthermore, genetic manipulation of key members of the m6A pathway, such as Mettl14, a component of the m6A methyltransferase complex, or the m6A reader Ythdf1, greatly attenuated the regenerative response in the PNS. Although a full description of the contribution of the epi-transcriptome to tissue regeneration is out of the scope of the present chapter, these results reveal the complexity at the basis of the regulation transcriptional program that needs to be activated for proper axonal regeneration in the PNS. For a discussion on how posttranscriptional RNA modifications affect transcriptional programs the reader can refer to excellent reviews in the field.127, 128

14.6 THERAPEUTIC INTERVENTIONS TARGETING THE EPIGENOME TO PROMOTE AXONAL REGENERATION The work discussed so far demonstrates that although the mammalian CNS is not capable of mounting a complete regenerative response in response to nerve lesion, it is not passive in its response to axonal damage. We therefore argue that pharmacological modulation of the epigenome can be explored as a way to potentiate axon regrowth, either alone or as part of combined treatments, in the regenerationresistant CNS. Recently, epigenetic approaches have been revealed as a means to increase either neuronal survival or axon regeneration in a variety of neurological disorders, such as traumatic brain injury, SCI, or stroke. Several epigenetic drugs have now been tested for their ability in regulating neuronal growth and survival in rodent models. For instance, treatment with the pan-HDACi trichostatin A (TSA) promotes axonal growth in primary rat neurons through a mechanism involving transcriptional activation.78 HDACis (TSA, MS-275) also promote regeneration in vivo in a mouse model of spinal cord injury by inducing transcription of RAGs.46 Moreover, valproic acid (VPA) attenuates inflammation and promotes axon regeneration and functional outcome after spinal cord injury,129, 130 while it plays a neuroprotective role in injured RGCs.131 On the other hand, global changes in the DNA methylome through pharmacological inhibition of DNMTs by RG108 has a neuroprotective role, preventing axotomy-induced cell death of spinal motor neurons,132 but its role in axonal regeneration has not been addressed yet. Changes in the epigenome can be also obtained through dietary interventions. For instance, interfering with the folate cycle through supplementation with folic acid

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leads to methylation of specific genes and promotes regeneration in combined spinal cord and sciatic nerve injuries.64 In addition to pharmacological modulation of the epigenome, manipulation of noncoding RNAs, and in particular miRNA levels, is now arising as an attractive strategy for axonal regeneration. However, amongst the main difficulties of RNA-based therapies for stimulating regeneration is the low stability of RNA molecules in vivo and the difficulty of obtaining targeted delivery of the therapeutic miRNAs/ antagomir. As such, many labs are now working on optimizing the delivery of miRNA mimics and antagomir into the nervous system. Currently, efforts are focused on optimizing adenoassociated viruses for safe and efficient in vivo delivery and on the development of novel RNA-based nanotherapies able to reach the nervous system.133 Finally, it was recently shown that the age-dependent decline of regeneration in the PNS correlates with the misfunction of Schwann cells. In the PNS, Schwann cells contribute to the regenerative response by contributing to myelin clearance and macrophage recruitment to the site of lesion.134 Therefore strategies to extend their function would likely complement the specific targeting of the neuronal compartment. Given that epigenetic interventions aimed at stimulating axonal regeneration are systemic, protocols should be designed considering the effect they may have in this and other supporting populations, especially in the regeneration-incompetent CNS.

14.7 CONCLUSIONS AND PERSPECTIVES Within this chapter, we aimed to give an overview of the intrinsic and extrinsic mechanisms of axonal regeneration, with a focus on the fundamental role of the epigenome in this process. We highlighted the common and diverse mechanisms that take place upon injury to the CNS and PNS, identifying key factors that may contribute to the impaired regeneration potential of the CNS. Although we are still far from a clinical translation of these results, the plasticity of the epigenome places it as an attractive target for therapeutic interventions. A better understanding of both the mechanisms that are activated in response to peripheral damage and the epigenetic barriers to tissue regeneration in the CNS is fundamental to the design of combined protocols for a variety of neurological disorders. In addition, work done on other tissues and on organisms with a higher regenerative capability may also help us to identify new molecules and pathways for targeted interventions. Finally, a note of caution should be placed as pharmacological interventions aimed at manipulating the epigenome are systemic and as such have the potential to target other tissues and organs. Yet, preclinical and clinical trials using epigenetic drugs for a variety of conditions have shown that epigenetic interventions are usually well tolerated and have few collateral effects. It has been argued that this is due to the fact that epigenetic treatments target cells with a more plastic epigenome, such as stem and tumor cells, or cells present in a regenerative microenvironment. For instance, as discussed above, an epigenetic response is also elicited in Schwann cells upon peripheral lesions, and such a response contributes to axonal regeneration in the PNS. Therefore protocols aimed at increasing axon regeneration in the CNS should take this in account and consider the effect they may have on other tissue resident populations, such as glia cells.

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LIST OF ABBREVIATIONS 5hmC 5meC CNS CSPG DMR DNMT DRG ERK HAT HDAC HDACi KSPG lncRNA MeDIP miRNA NAD NGS ON PCAF PNS PRC RAG RGC SAM SC SCI TDG TET TSA VPA

five-hydroxymethylcytosine five-methylcytosine central nervous system chondroitin sulfate proteoglycans differentially methylated region DNA methyltransferase dorsal root ganglia extracellular signal-related kinase histone acetyltransferases histone deacetylase HDAC inhibitor keratin-sulfate proteoglycan long noncoding RNA methylated DNA immunoprecipitation microRNA nicotinamide adenine dinucleotide next-generation sequencing optic nerve p300/CBP-associated factor peripheral nervous system Polycomb repressive complex regeneration-associated gene retinal ganglion cell S-adenosine methionine spinal cord spinal cord injury thymidine DNA glycosylase Tet-eleven translocation trichostatin A valproic acid

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FURTHER READING 135. Hanz S, Fainzilber M. Retrograde signaling in injured nerve—the axon reaction revisited. J Neurochem. 2006;99(1):13–19.