Secretion of EGF-like domain of heregulinβ promotes axonal growth and functional recovery of injured sciatic nerve

Neuregulin 1 (NRG1) and epidermal growth factor receptor (ErbB) signaling pathways control Schwann cells during axonal regeneration in an injured peripheral nervous system. We investigated whether a persistent supply of recombinant NRG1 to the injury site could improve axonal growth and recovery of sensory and motor functions in rats during nerve regeneration. We generated a recombinant adenovirus expressing a secreted form of EGF-like domain from Heregulinβ (sHRGβE-Ad). This virus, sHRGβE-Ad allowed for the secretion of 30-50 ng of small sHRGβE peptides per 10^7–8 virus particle outside cells and was able to phosphorylate ErbB receptors. Transduction of the concentrated sHRGβE-Ad into an axotomy model of sciatic nerve damage caused an effective promotion of nerve regeneration, as shown by histological features of the axons and Schwann cells, as well as increased expression of neurofilaments, GAP43 and S100 in the distal stump of the injury site. This result is consistent with longer axon lengths and thicker calibers observed in the sHRGβE-Ad treated animals. Furthermore, sensory and motor functions were significantly improved in sHRGβE-Ad treated animals when evaluated by a behavioral test. These results suggest a therapeutic potential for sHRGβE-Ad in treatment of peripheral nerve injury.     

Axonal Regeneration after Sciatic Nerve Lesion Is Delayed but Complete in GFAP- and Vimentin-Deficient Mice

Peripheral axotomy of motoneurons triggers Wallerian degeneration of injured axons distal to the lesion, followed by axon regeneration. Centrally, axotomy induces loss of synapses (synaptic stripping) from the surface of lesioned motoneurons in the spinal cord. At the lesion site, reactive Schwann cells provide trophic support and guidance for outgrowing axons. The mechanisms of synaptic stripping remain elusive, but reactive astrocytes and microglia appear to be important in this process. We studied axonal regeneration and synaptic stripping of motoneurons after a sciatic nerve lesion in mice lacking the intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin, which are upregulated in reactive astrocytes and Schwann cells. Seven days after sciatic nerve transection, ultrastructural analysis of synaptic density on the somata of injured motoneurons revealed more remaining boutons covering injured somata in GFAP^–/–Vim^–/– mice. After sciatic nerve crush in GFAP^–/–Vim^–/– mice, the fraction of reinnervated motor endplates on muscle fibers of the gastrocnemius muscle was reduced 13 days after the injury, and axonal regeneration and functional recovery were delayed but complete. Thus, the absence of GFAP and vimentin in glial cells does not seem to affect the outcome after peripheral motoneuron injury but may have an important effect on the response dynamics.     

Leukemia inhibitory factor regulates Schwann cell proliferation and migration and affects peripheral nerve regeneration

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that stimulates neuronal development and survival. Our previous study has demonstrated that LIF mRNA is dysregulated in the peripheral nerve segments after nerve injury. Here, we show that LIF protein is abundantly expressed in Schwann cells after rat sciatic nerve injury. Functionally, suppressed or elevated LIF increases or decreases the proliferation rate and migration ability of Schwann cells, respectively. Morphological observations demonstrate that in vivo application of siRNA against LIF after peripheral nerve injury promotes Schwann cell migration and proliferation, axon elongation, and myelin formation. Electrophysiological and behavior assessments disclose that knockdown of LIF benefits the function recovery of injured peripheral nerves. Differentially expressed LIF affects the metabolism of Schwann cells and negatively regulates ERFE (Erythroferrone). Collectively, our observations reveal the essential roles for LIF in regulating the proliferation and migration of Schwann cells and the regeneration of injured peripheral nerves, discover ERFE as a downstream effector of LIF, and extend our understanding of the molecular mechanisms underlying peripheral nerve regeneration.Subject terms: Cell migration, Regeneration and repair in the nervous system     

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.     

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.     

RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

This study shows that p90 ribosomal S6 kinase 1 (RSK1) responds differentially to nerve injury in the peripheral and central nervous systems, and identifies it as a crucial regulator of axonal regeneration; mechanistically, RSK1 preferentially induces the synthesis of regeneration-related proteins via the RSK1-eEF2K-eEF2 axis.     

Enhanced axon outgrowth and improved long‐distance axon regeneration in sprouty2 deficient mice

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal‐regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2^+/? neuron cultures, whereas homozygous Spry2^?/? neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2^+/? mice recovered faster in motor but not sensory testing paradigms (Spry2^?/? mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP‐43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2^?/? mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4‐positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long‐distance axon regeneration in injured peripheral nerves. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 217–231, 2015     

Beneficial Effect of Metformin on Nerve Regeneration and Functional Recovery After Sciatic Nerve Crush Injury in Diabetic Rats

Neuroprotective effects of metformin have been increasingly recognized in both diabetic and non-diabetic conditions. Thus far, no information has been available on the potential beneficial effects of metformin on peripheral nerve regeneration in diabetes mellitus. The present study was designed to investigate such a possibility. Diabetes was established by a single injection of streptozotocin at 50 mg/kg in rats. After sciatic nerve crush injury, the diabetic rats were intraperitoneally administrated daily for 4 weeks with metformin (30, 200 and 500 mg/kg), or normal saline, respectively. The axonal regeneration was investigated by morphometric analysis and retrograde labeling. The functional recovery was evaluated by electrophysiological studies and behavioral analysis. It was found that metformin significantly enhanced axonal regeneration and functional recovery compared to saline after sciatic nerve injury in diabetic rats. In addition, metformin at 200 and 500 mg/kg showed better performance than that at 30 mg/kg. Taken together, metformin is capable of promoting nerve regeneration after sciatic nerve injuries in diabetes mellitus, highlighting its therapeutic values for peripheral nerve injury repair in diabetes mellitus.     

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.     

Mice lacking glutamate carboxypeptidase II are protected from peripheral neuropathy and ischemic brain injury

Excessive glutamate release is associated with neuronal damage. A new strategy for the treatment of neuronal injury involves inhibition of the neuropeptidase glutamate carboxypeptidase II (GCP II), also known as N-acetylated alpha-linked acidic dipeptidase. GCP II is believed to mediate the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to glutamate and N-acetyl-aspartate, and inhibition of NAAG peptidase activity (by GCP II and other peptidases) is neuroprotective. Mice were generated in which the Folh1 gene encoding GCP II was disrupted (Folh1-/- mice). No overt behavioral differences were apparent between Folh1-/- mice and wild-type littermates, with respect to their overall performance in locomotion, coordination, pain threshold, cognition and psychiatric behavioral paradigms. Morphological analysis of peripheral nerves, however, showed significantly smaller axons (reduced myelin sheaths and axon diameters) in sciatic nerves from Folh1-/- mice. Following sciatic nerve crush, Folh1-/- mice suffered less injury and recovered faster than wild-type littermates. In a model of ischemic injury, the Folh1-/- mice exhibited a significant reduction (p < 0.05) in infarct volume compared with their wild-type littermates when subjected to middle cerebral artery occlusion, a model of stroke. These findings support the hypothesis that GCP II inhibitors may represent a novel treatment for peripheral neuropathies as well as stroke.     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

Synergistic Effects of Bone Mesenchymal Stem Cells and Chondroitinase ABC on Nerve Regeneration After Acellular Nerve Allograft in Rats

This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague–Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

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.