Signaling the pathway to regeneration

Robust axon regeneration occurs after peripheral nerve injury through coordinated activation of a genetic program and local intracellular signaling cascades. Although regeneration-associated genes are being identified with increasing frequency, most aspects of regeneration-associated intracellular signaling remain poorly understood. Two independent studies now report that upregulation of cAMP is a component of the PNS regeneration program that can be exploited to enhance axon regeneration through the normally inhibitory CNS environment.     

Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration

Inactivation of glycogen synthase kinase 3 (GSK3) has been shown to mediate axon growth during development and regeneration. Phosphorylation of GSK3 by the kinase Akt is well known to be the major mechanism by which GSK3 is inactivated. However, whether such regulatory mechanism of GSK3 inactivation is used in neurons to control axon growth has not been directly studied. Here by using GSK3 mutant mice, in which GSK3 is insensitive to Akt-mediated inactivation, we show that sensory axons regenerate normally in vitro and in vivo after peripheral axotomy. We also find that GSK3 in sensory neurons of the mutant mice is still inactivated in response to peripheral axotomy and such inactivation is required for sensory axon regeneration. Lastly, we provide evidence that GSK3 activity is negatively regulated by PI3K signaling in the mutant mice upon peripheral axotomy, and the PI3K–GSK3 pathway is functionally required for sensory axon regeneration. Together, these results indicate that in response to peripheral nerve injury GSK3 inactivation, regulated by an alternative mechanism independent of Akt-mediated phosphorylation, controls sensory axon regeneration.     

Neurotrophic factors and axonal growth

Neuronal morphological differentiation is regulated by numerous polypeptide growth factors (neurotrophic factors). Recently, significant progress has been achieved in clarifying the roles of neurotrophins as well as glial cell line-derived neurotrophic factor family members in peripheral axon elongation during development. Additionally, advances have been made in defining the signal transduction mechanisms employed by these factors in mediating axon morphological responses. Several studies addressed the role of neurotrophic factors in regenerative axon growth and suggest that signaling mechanisms in addition to those triggered by receptor tyrosine kinases may be required for successful peripheral nervous system regeneration. Finally, recent investigations demonstrate that neurotrophic factors can enhance axon growth after spinal cord injuries.     

中枢神经损伤后影响轴突再生的因素

与周围神经不同 ,成年哺乳动物中枢神经损伤后轴突不能再生 ,往往造成不可逆的功能丧失。影响再生的原因相当复杂 ,胶质瘢痕形成、神经营养因子缺乏及存在诸多的抑制性因子等。本文就一些影响中枢神经再生的因子从其结构、分布、功能及可能的作用机制诸方面作一综述     

Inhibition of PTEN activity promotes IB4‐positive sensory neuronal axon growth

Traumatic nerve injuries have become a common clinical problem, and axon regeneration is a critical process in the successful functional recovery of the injured nervous system. In this study, we found that peripheral axotomy reduces PTEN expression in adult sensory neurons; however, it did not alter the expression level of PTEN in IB4‐positive sensory neurons. Additionally, our results indicate that the artificial inhibition of PTEN markedly promotes adult sensory axon regeneration, including IB4‐positive neuronal axon growth. Thus, our results provide strong evidence that PTEN is a prominent repressor of adult sensory axon regeneration, especially in IB4‐positive neurons.     

Spinal nerve segmentation in the chick embryo: analysis of distinct axon-repulsive systems

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Molecular and cellular aspects of nerve regeneration

Injury of an axon leads to at least four independent events, summarized in Figure 1: first, deprivation of the nerve cell body from target-derived or mediated substances, which leads to a derepressed or a permissive state; second, disruption of anterograde transport, with a resultant accumulation of anterogradely transported molecules; third, environmental response with possible consequent changes in constituents of the extracellular matrix and substances secreted from the surrounding cells; and fourth, appearance of growth inhibitors and modified protease activity. It seems that the first three of these events are obligatory, but not sufficient, i.e., they lead to a growth state only if the cell body is able to respond to the injury-induced signals from the environment (a and b). The regenerative state is characterized by alterations in protein synthesis and axonal transport and by sprouting activity. The subsequent elongation of the growing fibers depends on a continuous supply of appropriate growth factors. These factors are presumably anchored to the appropriate extracellular matrix that serves as a substratum for elongating fibers. It should be mentioned that the proliferating nonneuronal cells have a conducive effect on regeneration by forming a scaffold for the growing fibers. Accordingly, the lack of regeneration may stem from a deficiency in the ability of glial cells to provide the appropriate soluble components or from insufficient formation of extracellular matrix. In this respect, one may consider regeneration of an injured axon as a process which involves regeneration of both the nonneuronal cells and the supported axons. The regeneration of glial cells may fulfill the rules which are applied to regeneration of any other proliferating tissue. Furthermore, the processes of regeneration in the axon and the glial cells are mutually dependent. Perhaps the triggering factors provided by the nonneuronal cells affect the nonneuronal cells themselves by modulating their postlesion gliosis and thereby inducing their appropriate activation. In such a case, regeneration of nonneuronal cells may resemble an autocrine type of regulation that exists also during ontogeny. The growth regulation is shifted back to the paracrine type upon neuronal maturation or cessation of axonal growth. When the elongating fibers reach the vicinity of the target organ, they are under the influence of the target-derived factors, which guide the fibers and eventually cease their elongation.     

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca²⁺) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.     

AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

Axon regeneration in the injured adult CNS is reportedly inhibited by myelin-derived inhibitory molecules, after binding to a receptor complex comprised of the Nogo-66 receptor (NgR1) and two transmembrane co-receptors p75/TROY and LINGO-1. However, the post-injury expression pattern for LINGO-1 is inconsistent with its proposed function. We demonstrated that AMIGO3 levels were significantly higher acutely than those of LINGO-1 in dorsal column lesions and reduced in models of dorsal root ganglion neuron (DRGN) axon regeneration. Similarly, AMIGO3 levels were raised in the retina immediately after optic nerve crush, whilst levels were suppressed in regenerating optic nerves, induced by intravitreal peripheral nerve implantation. AMIGO3 interacted functionally with NgR1-p75/TROY in non-neuronal cells and in brain lysates, mediating RhoA activation in response to CNS myelin. Knockdown of AMIGO3 in myelin-inhibited adult primary DRG and retinal cultures promoted disinhibited neurite growth when cells were stimulated with appropriate neurotrophic factors. These findings demonstrate that AMIGO3 substitutes for LINGO-1 in the NgR1-p75/TROY inhibitory signalling complex and suggests that the NgR1-p75/TROY-AMIGO3 receptor complex mediates myelin-induced inhibition of axon growth acutely in the CNS. Thus, antagonizing AMIGO3 rather than LINGO-1 immediately after CNS injury is likely to be a more effective therapeutic strategy for promoting CNS axon regeneration when combined with neurotrophic factor administration.     

Role of macrophages in peripheral nerve degeneration and repair.

A cut or crush injury to a peripheral nerve results in the degeneration of that portion of the axon isolated from the cell body. The rapid degeneration of this distal segment was for many years believed to be a process intrinsic to the nerve. It was believed that Schwann cells both phagocytosed degenerating axons and myelin sheaths and also provided growth factors to promote regeneration of the damaged axons. In recent years, it has become apparent that the degenerating distal segment is invaded by monocytes from the blood. We will review the evidence that these recruited macrophages play a role in both degeneration and regeneration of peripheral nerve axons after injury and consider whether the slow degeneration and poor monocyte recruitment in the central nervous system may contribute to the poor regeneration there.     

Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin

Functional recovery from cutaneous injury requires not only the healing and regeneration of skin cells but also reinnervation of the skin by somatosensory peripheral axon endings. To investigate how sensory axon regeneration and wound healing are coordinated, we amputated the caudal fins of zebrafish larvae and imaged somatosensory axon behavior. Fin amputation strongly promoted the regeneration of nearby sensory axons, an effect that could be mimicked by ablating a few keratinocytes anywhere in the body. Since injury produces the reactive oxygen species hydrogen peroxide (H(2)O(2)) near wounds, we tested whether H(2)O(2) influences cutaneous axon regeneration. Exposure of zebrafish larvae to sublethal levels of exogenous H(2)O(2) promoted growth of severed axons in the absence of keratinocyte injury, and inhibiting H(2)O(2) production blocked the axon growth-promoting effects of fin amputation and keratinocyte ablation. Thus, H(2)O(2) signaling helps coordinate wound healing with peripheral sensory axon reinnervation of the skin.     

Role of Non-coding RNAs in Axon Regeneration after Peripheral Nerve Injury

Peripheral nerve injury (PNI) may lead to disability and neuropathic pain, which constitutes a substantial economic burden to patients and society. It was found that the peripheral nervous system (PNS) has the ability to regenerate after injury due to a permissive microenvironment mainly provided by Schwann cells (SCs) and the intrinsic growth capacity of neurons; however, the results of injury repair are not always satisfactory. Effective, long-distance axon regeneration after PNI is achieved by precise regulation of gene expression. Numerous studies have shown that in the process of peripheral nerve damage and repair, differential expression of non-coding RNAs (ncRNAs) significantly affects axon regeneration, especially expression of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In the present article, we review the cellular and molecular mechanisms of axon regeneration after PNI, and analyze the roles of these ncRNAs in nerve repair. In addition, we discuss the characteristics and functions of these ncRNAs. Finally, we provide a thorough perspective on the functional mechanisms of ncRNAs in nervous injury repair, and explore the potential these ncRNAs offer as targets of nerve injury treatment.     

Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.