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.     

Establishment of axon regeneration regulatory network and the role of low intensity pulsed ultrasound in the network

ObjectiveTo establish an axon regeneration regulatory network for optimal selection, and explore the role of low intensity pulsed ultrasound in the network.MethodsThe axon regeneration regulatory network involving axon regeneration-related proteins NGF, BDNF and PirB was constructed by using GO and KEGG. The maximum possible pathway acting on axon regeneration was screened by Bayesian network theory. The node of low - intensity pulsed ultrasound in NGF - involved axon regeneration network was complemented by combining literature methods.ResultsThe NGF, BDNF and PirB-involved axonal regeneration regulatory pathway was successfully constructed. The low intensity pulsed ultrasound played a role in axon regeneration by acting on ERK1/2-CREB pathway and GSK-3β. NGF-TrKA-Rap1-ERK1/2-CREB-Bcl-2 was optimized as optimal pathway by Bayesian theory.ConclusionThe regulatory pathway of axon regeneration involving nerve growth related factors and low intensity pulsed ultrasound was initially established, which provided a theoretical basis for further study of axon regeneration, and also new ideas for action of low intensity pulsed ultrasound on axon regeneration regulatory pathway.     

C. elegans as a genetic model to identify novel cellular and molecular mechanisms underlying nervous system regeneration

Research into conditions that improve axon regeneration has the potential to open a new door for treatment of brain injury caused by stroke and neurodegenerative diseases of aging, such as Alzheimer, by harnessing intrinsic neuronal ability to reorganize itself. Elucidating the molecular mechanisms of axon regeneration should shed light on how this process becomes restricted in the postnatal stage and in CNS and therefore could provide therapeutic targets for developing strategy to improve axon regeneration in adult CNS. In this review, we first discuss the general view about nerve regeneration and the advantages of using C. elegans as a model system to study axon regeneration. We then compare the conserved regeneration patterns and molecular mechanisms between C. elegans and vertebrates. Lastly, we discuss the power of femtosecond laser technology and its application in axon regeneration research.     

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.     

C. elegans as a genetic model to identify novel cellular and molecular mechanisms underlying nervous system regeneration

Research into conditions that improve axon regeneration has the potential to open a new door for treatment of brain injury caused by stroke and neurodegenerative diseases of aging, such as Alzheimer, by harnessing intrinsic neuronal ability to reorganize itself. Elucidating the molecular mechanisms of axon regeneration should shed light on how this process becomes restricted in the postnatal stage and in the CNS and therefore could provide therapeutic targets for developing strategies to improve axon regeneration in the adult CNS. In this review, we first discuss the general view about nerve regeneration and the advantages of using C. elegans as a model system to study axon regeneration. We then compare the conserved regeneration patterns and molecular mechanisms between C. elegans and vertebrates. Lastly, we discuss the power of femtosecond laser technology and its application in axon regeneration research.Key words: axon regeneration, C. elegans, genetics, femtosecond laser, neuronal circuits     

Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.     

Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

The molecular and cellular mechanisms that allow adult-stage neurons to regenerate following damage are poorly understood. Recently, axons of motoneurons and mechanosensory neurons in adult C. elegans were found to regrow after being snipped by femtosecond laser ablation. Here, we explore the molecular determinants of adult-stage axon regeneration using the AVM mechanosensory neurons. The first step in AVM axon development is a pioneer axonal projection from the cell body to the ventral nerve cord. We show that regeneration of the AVM axon to the ventral nerve cord lacks the deterministic precision of initial axon development, requiring competition and pruning of unwanted axon branches. Nevertheless, axons of injured AVM neurons regrow to the ventral nerve cord with over 60% reliability in adult animals. In addition, in contrast to initial development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but independent of UNC-129/TGF-beta repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes heavily dependent on UNC-34/Ena and CED-10/Rac actin regulators. Thus, C. elegans may be used as a genetic system to characterize novel cellular and molecular mechanisms underlying adult-stage nervous system regeneration.     

Sex differences in the effectiveness of treadmill training in enhancing axon regeneration in injured peripheral nerves

Exercise in the form of daily treadmill training results in significant enhancement of axon regeneration following peripheral nerve injury. Because androgens are also linked to enhanced axon regeneration, we wanted to investigate whether sex differences in the effect of treadmill training might exist. The common fibular nerves of thy-1-YFP-H mice were cut and repaired with a graft of the same nerve from a strain-matched wild-type donor mouse. Animals were treated with one of two daily treadmill training paradigms: slow continuous walking for 1 h or four higher intensity intervals of 2 min duration separated by 5-min rest periods. Training was begun on the third day following nerve injury and continued 5 days per week for 2 weeks. Effects on regeneration were evaluated by measuring regenerating axon profile lengths in optical sections through the repair sites and grafts at the end of the training period. No sex differences were found in untrained control mice. Continuous training resulted in significant enhancement of axon regeneration only in males. No effect was found in females or in castrated males. Interval training was effective in enhancing axon regeneration only in females and not in intact males or castrated males. Untrained females treated with the aromatase inhibitor, anastrozole, had significant enhancement of axon regeneration without increasing serum testosterone levels. Two different mechanisms exist to promote axon regeneration in a sex-dependent manner. In males treadmill training uses testicular androgens. In females, a different cellular mechanism for the effect of treadmill training must exist.     

Induction of Growth Cone Formation by Transient and Localized Increases of Intracellular Proteolytic Activity

The formation of a growth cone at the tip of a transected axon is a crucial step in the subsequent regeneration of the amputated axon. During this process, the transected axon is transformed from a static segment into a motile growth cone. Despite the importance of this process for regeneration of the severed axon, little is known about the mechanisms underlying this transformation.     

Axon regeneration: Vaccinating against spinal cord injury

Myelin is a potent inhibitor of axon regeneration, but has been viewed as just one of many factors that prevent regeneration after injury. So it comes as a surprise that immunization against myelin has been found to allow extensive axon regeneration after injury, without apparent autoimmune-induced demyelination.     

Repulsive factors and axon regeneration in the CNS

During the past year, a major advance in the study of axon regeneration was the molecular cloning of Nogo. The expression of Nogo protein by CNS myelin may be a major factor in the failure of CNS axon regeneration. The effect of disrupting Nogo-dependent axon inhibition can now be studied conclusively. In related work, immunization with a Nogo-containing CNS myelin preparation was shown to promote regeneration and dramatic functional recovery after spinal cord trauma.     

Signaling mechanisms of the myelin inhibitors of axon regeneration

One of the major obstacles to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that are associated with myelin. Recent studies have identified several major myelin-associated inhibitors along with the relevant signaling molecules. Such advances have not only enhanced our understanding of the signaling mechanisms that are involved in the inhibition of axon regeneration in the adult CNS but also allowed us to assess the therapeutic potential of blocking these inhibitory influences to promote axon regeneration.     

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.     

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.     

Attenuation of Axonal Degeneration by Calcium Channel Inhibitors Improves Retinal Ganglion Cell Survival and Regeneration After Optic Nerve Crush

Axonal degeneration is one of the initial steps in many traumatic and neurodegenerative central nervous system (CNS) disorders and thus a promising therapeutic target. A focal axonal lesion is followed by acute axonal degeneration (AAD) of both adjacent axon parts, before proximal and distal parts follow different degenerative fates at later time points. Blocking calcium influx by calcium channel inhibitors was previously shown to attenuate AAD after optic nerve crush (ONC). However, it remains unclear whether the attenuation of AAD also promotes consecutive axonal regeneration. Here, we used a rat ONC model to study the effects of calcium channel inhibitors on axonal degeneration, retinal ganglion cell (RGC) survival, and axonal regeneration, as well as the molecular mechanisms involved. Application of calcium channel inhibitors attenuated AAD after ONC and preserved axonal integrity as visualized by live imaging of optic nerve axons. Consecutively, this resulted in improved survival of RGCs and improved axonal regeneration at 28 days after ONC. We show further that calcium channel inhibition attenuated lesion-induced calpain activation in the proximity of the crush and inhibited the activation of the c-Jun N-terminal kinase pathway. Pro-survival signaling via Akt in the retina was also increased. Our data thus show that attenuation of AAD improves consecutive neuronal survival and axonal regeneration and that calcium channel inhibitors could be valuable tools for therapeutic interventions in traumatic and degenerative CNS disorders.     

rab-27 acts in an intestinal pathway to inhibit axon regeneration in C. elegans

Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, KPC3/aex-5, and the neuropeptide NLP-40, and re-expression of these genes in the intestine of mutant animals is sufficient to restore normal regeneration success. Additionally, NPDC1/cab-1 and SNAP25/aex-4 genetically interact with rab-27 in the context of axon regeneration inhibition. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.