Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of ΔFosB. In hippocampus of rat brain, expression of ΔFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury. Published in 2004.     

Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties.

Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known beta-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2-Cys130, Cys16-Cys88 and Cys42-Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0-5000 pg. mL-1), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

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.     

Regeneration of optic nerve fibers of adult mammals

The pathway from the retina to the brain in mammals provides a well-defined model system for investigation of not only surviving axotomy but also axonal regeneration of injured neurons. Here I introduce our recent works on axonal regeneration in the optic nerve (OpN) of adult cats. Fibers of retinal ganglion cells (RGCs) extend beyond the crush site of OpN with injections of a macrophage stimulator (oxidized galectin-1) or a Rho kinase (ROCK) inhibitor (Y-39983 or Y-27632) while axonal extension is blocked with injection of saline. Elongation of crushed optic fibers, however, is slowed after 2 weeks. Transplantation of peripheral nerve makes RGCs regenerate their transected axons into a graft but regenerated fibers extend only a few mm in the brain. Effectiveness of combination of the drugs and treatments has to be verified in future.     

Melanocortin receptor 4 is induced in nerve-injured motor and sensory neurons of mouse

We previously identified melanocortin receptor 4 (MC4R) in a search for genes associated with hypoglossal nerve regeneration. As melanocortins promote nerve regeneration after axonal injury, we investigated whether MC4R functions as a key receptor for peripheral nerve regeneration. In situ hybridization revealed that MC4R mRNA is induced in mouse hypoglossal motor neurons after axonal injury, whereas mRNAs for MC1R, MC2R, MC3R, and MC5R are not expressed either before or after nerve injury. This result was confirmed by RT-PCR. The level of MC4R mRNA expression increased significantly from day 3 after axotomy, reached a peak on day 5, and decreased to the control level on day 14. Similar induction of MC4R was observed in axotomized mouse dorsal root ganglia (DRGs). MC4R mRNA expression was induced exclusively among the MCR family in the L4-6 DRG after sciatic nerve injury. We further examined whether alpha-melanocortin stimulating hormone (alpha-MSH) promotes neurite elongation via MC4R. In mouse DRG neuron culture, alpha-MSH significantly promoted neurite outgrowth at a concentration of 10(-8) mol/L. This neurite-elongation effect was entirely inhibited by the addition of a selective MC4R blocker, JKC-363. Therefore, it is concluded that alpha-MSH could stimulate neurite elongation via MC4R in DRG neurons. The present results suggest that induction of MC4R is crucial for motor and sensory neurons to regenerate after axonal injury.     

FLRT3, a cell surface molecule containing LRR repeats and a FNIII domain, promotes neurite outgrowth

The mature peripheral nervous system has the ability to survive and to regenerate its axons following axonal injury. After nerve injury, the distal axonal and myelin segment undergoes dissolution and absorption by the surrounding cellular environment, a process called Wallerian degeneration. Using cDNA microarrays, we isolated FLRT3 as one of the up-regulated genes expressed in the distal segment of the sciatic nerve 7 days after transection relative to those of the intact sciatic nerve. FLRT3 is a putative type I transmembrane protein containing 10 leucine-rich repeats, a fibronectin type III domain, and an intracellular tail. The neurons plated on CHO cells expressing FLRT3 extended significantly longer neurites than those plated on wild-type CHO cells, demonstrating that FLRT3 promotes neurite outgrowth. FLRT3 mRNA was especially abundant in the basal ganglia, the granular layer of cerebellum, and the hippocampus, except the CA1 region in the adult rat brain. Thus, FLRT3 may contribute to regeneration following axonal injury.     

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     

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.     

Temporal-Spatial Expressions of Spy1 in Rat Sciatic Nerve After Crush

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post-injured sciatic nerve which was enriched in non-myelinating Schwann cells using cDNA microarrays. The number of up- and down-regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up-regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down-regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co-receptor for glial cell line-derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

Dihydrotestosterone Treatment Accelerates Autograft Reversal Sciatic Nerve Regeneration in Rats

Neuroactive steroids such as progesterone, testosterone, and their derivatives have been widely studied for their neuroprotective roles in the nervous system. Autologous nerve transplantation is considered as the gold standard repair technique when primary suture is impossible; nevertheless, this method is far from ideal. In this study, we aimed to explore the impact of dihydrotestosterone (DHT), a 5α-reduced derivative of testosterone, on the recovery of peripheral nerve injury treated with autologous nerve transplantation. Sprague–Dawley rats were subjected to a 10-mm right side sciatic nerve reversed autologous nerve transplantation and randomly divided into groups that received DHT or DHT?+?flutamide (an androgen receptor blocker) daily for 8 weeks after operation. Our results demonstrated that DHT could speed up the rate of axonal regeneration and increase the expression of myelin protein zero (P0) in autograft reversal sciatic nerves. Thus, our study provided new insights into improving the prognosis of patients with long gap peripheral nerve defects.     

Selective induction of DeltaFosB in the brain after transient forebrain ischemia accompanied by an increased expression of galectin-1, and the implication of DeltaFosB and galectin-1 in neuroprotection and neurogenesis

Transient forebrain ischemia causes selective induction of DeltaFosB, an AP-1 (activator protein-1) subunit, in cells within the ventricle wall or those in the dentate gyrus in the rat brain prior to neurogenesis, followed by induction of nestin, a marker for neuronal precursor cells, or galectin-1, a beta-galactoside sugar-binding lectin. The adenovirus-mediated expression of FosB or DeltaFosB induced expression of nestin, glial fibrillary acidic protein and galectin-1 in rat embryonic cortical cells. DeltaFosB-expressing cells exhibited a significantly higher survival and proliferation after the withdrawal of B27 supplement than the control or FosB-expressing cells. The decline in the DeltaFosB expression in the survivors enhanced the MAP2 expression. The expression of DeltaFosB in cells within the ventricle wall of the rat brain also resulted in an elevated expression of nestin. We therefore conclude that DeltaFosB can promote the proliferation of quiescent neuronal precursor cells, thus enhancing neurogenesis after transient forebrain ischemia.     

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.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Galectin-1beta, a natural monomeric form of galectin-1 lacking its six amino-terminal residues promotes axonal regeneration but not cell death

We previously identified a novel N-terminally processed form of galectin-1, galectin-1beta (Gal-1beta) whose expression was induced by DeltaFosB. In the present study, the biochemical properties and biological functions of Gal-1beta were compared with the full-length form of galectin-1 (Gal-1alpha). We first purified recombinant mouse Gal-1alpha and beta (rmGal-1alpha, beta) to near homogeneity. The rmGal-1alpha exists as a monomer under oxidized conditions and forms a dimer under reduced conditions, while the rmGal-1beta exists as a monomer regardless of redox conditions. The affinity of rmGal-1beta to beta-lactose was approximately two-fold lower than that of rmGal-1alpha under reduced conditions. The viability of Jurkat cells efficiently decreased when they were exposed to rmGal-1alpha, however, rmGal-1beta barely induced such a reduction. In contrast, both rmGal-1alpha and rmGal-1beta exhibited an equivalent capacity to promote axonal regeneration from the dorsal root ganglion explants. Our results suggest that the biochemical properties of rmGal-1beta determine its biological functions.