Eml1 promotes axonal growth by enhancing αTAT1-mediated microtubule acetylation.

Microtubule stabilization is critical for axonal growth and regeneration, and many microtubule-associated proteins are involved in this process. In this study, we found that the knockdown of echinoderm microtubule-associated protein-like 1 (EML1) hindered axonal growth in cultured cortical and dorsal root ganglion neurons. We further revealed that EML1 facilitated the acetylation of microtubules and that the impairment of axonal growth due to EML1 inhibition could be restored by treatment with deacetylase inhibitors, suggesting that EML1 affected tubulin acetylation. Moreover, we verified an interaction between EML1 and the alpha-tubulin acetyltransferase 1, which is responsible for the acetylation of alpha-tubulin. We thus proposed that EML1 might regulate microtubule acetylation and stabilization via alpha-tubulin acetyltransferase 1 and then promote axon growth. Finally, we verified that the knockdown of EML1 in vivo also inhibited sciatic nerve regeneration. Our findings revealed a novel effect of EML1 on microtubule acetylation during axonal regeneration.

GIP attenuates neuronal oxidative stress by regulating glucose uptake in spinal cord injury of rat.

AIM: Glucose-dependent insulinotropic polypeptide (GIP) is a ligand of glucose-dependent insulinotropic polypeptide receptor (GIPR) that plays an important role in the digestive system. In recent years, GIP has been regarded as a hormone-like peptide to regulate the local metabolic environment. In this study, we investigated the antioxidant role of GIP on the neuron and explored the possible mechanism. METHODS: Cell counting Kit-8 (CCK-8) was used to measure cell survival. TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect apoptosis in vitro and in vivo. Reactive oxygen species (ROS) levels were probed with 2', 7'-Dichloro dihydrofluorescein diacetate (DCFH-DA), and glucose intake was detected with 2-NBDG. Immunofluorescence staining and western blot were used to evaluate the protein level in cells and tissues. Hematoxylin-eosin (HE) staining, immunofluorescence staining and tract-tracing were used to observe the morphology of the injured spinal cord. Basso-Beattie-Bresnahan (BBB) assay was used to evaluate functional recovery after spinal cord injury. RESULTS: GIP reduced the ROS level and protected cells from apoptosis in cultured neurons and injured spinal cord. GIP facilitated wound healing and functional recovery of the injured spinal cord. GIP significantly improved the glucose uptake of cultured neurons. Meanwhile, inhibition of glucose uptake significantly attenuated the antioxidant effect of GIP. GIP increased glucose transporter 3 (GLUT3) expression via up-regulating the level of hypoxia-inducible factor 1α (HIF-1α) in an Akt-dependent manner. CONCLUSION: GIP increases GLUT3 expression and promotes glucose intake in neurons, which exerts an antioxidant effect and protects neuronal cells from oxidative stress both in vitro and in vivo.

Promising application of a novel biomaterial, light chain of silk fibroin combined with NT3, in repairment of rat sciatic nerve defect injury.

Autologous nerve transplantation is the gold standard for treating peripheral nerve defects, but it is associated with defects such as insufficient donor and secondary injury. Artificial nerve guidance conduits (NGCs) are now considered promising alternatives for bridging long nerve gaps, although exploring new biomaterials to construct NGCs remains challenging. Silk fibroin (SF) has good biocompatibility and can self-assemble in aqueous solutions. However, the lack of proximal neurotrophic factors after nerve injury is a major concern, leading to incomplete nerve regeneration. In this study, NT-3, a neurotrophin that promotes neuronal survival and differentiation, was bound to the light chain of silk fibroin (FIBL) in two ways: one was directly bound to FIBL (FIBL-NT3) and the other was a polypeptides-linker (FIBL-Linker-NT3). The design aimed to take advantage of silk fiber's character of self-assembly of heavy-light chains and test whether a flexible linker with NT3 molecule is easy to be a NT3 dimer, the active form. In vitro studies indicated that FIBL-Linker-NT3 combined with SF membranes promoted axon growth in adult rat dorsal root ganglion (DRG) neurons. Then we tested if FIBL-Linker-NT3 could self-assemble with the SF heavy chain (SFH). DTT (Dithiothreitol) was used to break the disulfide bonds between the SF light and heavy chains, and the light-chain protein was removed via dialysis. SFH was assembled using FIBL-Linker-NT3, as evidenced by the western blotting results that showed a high molecular band corresponding to SFH-FIBL-Linker-NT3. Chitosan scaffolds have been identified to provide a suitable microenvironment, so a chitosan/SF-FIBL-Linker-NT3 conduit was also constructed. Nerve transplantation of this conduit was evaluated in vivo in a rat sciatic nerve defect model. Immunohistochemical assays showed that the chitosan/SF-FIBL-Linker-NT3 group was superior to the chitosan/PBS, SF, PBS + FIBL-Linker-NT3 groups in nerve regeneration. In addition, the chitosan/SF-FIBL-Linker-NT3 conduit-transplanted group exhibited better recovery in terms of neurite length, sciatic functional index value, sensitivity to heat, time on the rotarod, wet weight ratio, cross-sectional area, compound muscle action potential, number of myelin layers, and myelin thickness in the nerve. Taking together, our study identified that FIBL-Linker-NT3 could promote axonal growth and regeneration in vivo and in vitro and is a promising candidate biomaterial for artificial NGCs.

The activation of gastric inhibitory peptide/gastric inhibitory peptide receptor axis via sonic hedgehog signaling promotes the bridging of gapped nerves in sciatic nerve injury.

Schwann cells play an essential role in peripheral nerve regeneration by generating a favorable microenvironment. Gastric inhibitory peptide/gastric inhibitory peptide receptor (GIP/GIPR) axis deficiency leads to failure of sciatic nerve repair. However, the underlying mechanism remains elusive. In this study, we surprisingly found that GIP treatment significantly enhances the migration of Schwann cells and the formation of Schwann cell cords during recovery from sciatic nerve injury in rats. We further revealed that GIP and GIPR levels in Schwann cells were low under normal conditions, and significantly increased after injury demonstrated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Wound healing and Transwell assays showed that GIP stimulation and GIPR silencing could affect Schwann cell migration. In vitro and in vivo mechanistic studies based on interference experiment revealed that GIP/GIPR might promote mechanistic target of rapamycin complex 2 (mTORC2) activity, thus facilitating cell migration; Rap1 activation might be involved in this process. Finally, we retrieved the stimulatory factors responsible for GIPR induction after injury. The results indicate that sonic hedgehog (SHH) is a potential candidate whose expression increased upon injury. Luciferase and chromatin immunoprecipitation (ChIP) assays showed that Gli3, the target transcription factor of the SHH pathway, dramatically augmented GIPR expression. Additionally, in vivo inhibition of SHH could effectively reduce GIPR expression after sciatic nerve injury. Collectively, our study reveals the importance of GIP/GIPR signaling in Schwann cell migration, providing a therapeutic avenue toward peripheral nerve injury.

Histone acetyltransferase KAT2A modulates neural stem cell differentiation and proliferation by inducing degradation of the transcription factor PAX6.

Neural stem cells (NSCs) proliferation and differentiation rely on proper expression and posttranslational modification of transcription factors involved in the determination of cell fate. Further characterization is needed to connect modifying enzymes with their transcription factor substrates in the regulation of these processes. Here, we demonstrated that the inhibition of KAT2A, a histone acetyltransferase, leads to a phenotype of small eyes in the developing embryo of zebrafish, which is associated with enhanced proliferation and apoptosis of NSCs in zebrafish eyes. We confirmed that this phenotype is mediated by the elevated level of PAX6 protein. We further verified that KAT2A negatively regulates PAX6 at the protein level in cultured neural stem cells of rat cerebral cortex. We revealed that PAX6 is a novel acetylation substrate of KAT2A and the acetylation of PAX6 promotes its ubiquitination mediated by the E3 ligase RNF8 that facilitated PAX6 degradation. Our study proposes that KAT2A inhibition results in accelerated proliferation, delayed differentiation, or apoptosis, depending on the context of PAX6 dosage. Thus, the KAT2A/PAX6 axis plays an essential role to keep a balance between the self-renewal and differentiation of NSCs.

Fidgetin impacts axonal growth and branching in a local mTOR signal dependent manner.

Neurons require a constant increase in protein synthesis during axonal growth and regeneration. AKT-mTOR is a central pathway for mammalian cell survival and regeneration. Fidgetin (Fign) is an ATP-dependent microtubule (MT)-severing enzyme whose functions are associated with neurite outgrowth, axon regeneration and cell migration. Although most previous studies have indicated that depletion of Fign is involved in those biological activities by increasing labile MT mass, it remains unknown whether mTOR activation contributes to this process. Here, we showed that depletion of Fign enhanced p-mTOR/p-S6K activation, and the mTOR inhibitor Rapamycin inhibited axon outgrowth and p-rpS6 activation. We then investigated the effects of neuronal-specific Fign deletion in a rat spinal cord hemisection model by injecting syn-GFP Fign shRNA virus. BBB values revealed an improvement in functional recovery. The p-mTOR was activated along with neuronal Fign depletion. The syn-mCherry virus showed more sprouting neurites entering the injury region, which was confirmed by immunostaining GAP43 protein. Further, we showed that Fign siRNA treatment promoted axon outgrowth and branching, whose underlying mechanism was firstly attributed to local activation of the mTOR pathway, and increased MT dynamicity. Finally, considering L-leucine, promotes axonal growth and neuronal survival, we applied L-leucine with Fign depletion after spinal cord injury or in chondroitin sulfate proteoglycan inhibitory molecules. The phenomenon of synergistically augmented axon regeneration was observed. In summary, our results indicated a novel local mTOR pathway for fidgetin to impact axon growth and provided a combined strategy in SCI.

Cadherin-12 Regulates Neurite Outgrowth Through the PKA/Rac1/Cdc42 Pathway in Cortical Neurons.

Cadherins play an important role in tissue homeostasis, as they are responsible for cell-cell adhesion during embryogenesis, tissue morphogenesis, and differentiation. In this study, we identified Cadherin-12 (CDH12), which encodes a type II classical cadherin, as a gene that promotes neurite outgrowth in an in vitro model of neurons with differentiated intrinsic growth ability. First, the effects of CDH12 on neurons were evaluated via RNA interference, and the results indicated that the knockdown of CDH12 expression restrained the axon extension of E18 neurons. The transcriptome profile of neurons with or without siCDH12 treatment revealed a set of pathways positively correlated with the effect of CDH12 on neurite outgrowth. We further revealed that CDH12 affected Rac1/Cdc42 phosphorylation in a PKA-dependent manner after testing using H-89 and 8-Bromo-cAMP sodium salt. Moreover, we investigated the expression of CDH12 in the brain, spinal cord, and dorsal root ganglia (DRG) during development using immunofluorescence staining. After that, we explored the effects of CDH12 on neurite outgrowth in vivo. A zebrafish model of CDH12 knockdown was established using the NgAgo-gDNA system, and the vital role of CDH12 in peripheral neurogenesis was determined. In summary, our study is the first to report the effect of CDH12 on axonal extension in vitro and in vivo, and we provide a preliminary explanation for this mechanism.

The vascularization, innervation and myogenesis of early regenerated tail in Gekko japonicus.

Many species of lizards are capable of tail regeneration. There has been increased interest in the study of lizard tail regeneration in recent years as it is an amenable regeneration model for amniotes. In this study, Gekko japonicus was used as a model to investigate the initiation of vascularization, innervation and myogenesis during tail regeneration. We found that angiogenesis and axon regeneration occurred almost simultaneously within 4 days post amputation. The results showed that the endothelial cells of the original vasculature proliferated and extended into the blastema as capillary vessels, which inter-connected to form a capillary network. The nerve fibers innervated the regenerated tissue from the original spinal cord and dorsal root ganglia, and the fiber bundles increased during 14 days. Regenerating muscle tissues emerged 2 weeks after amputation. PAX3 and PAX7 expression were detected during myogenesis, with PAX7 showing a continuous increase in expression from day 3 until the day 14, whereas PAX3 reached a peak level on day 10 day post amputation, and then declined quickly to level as normal control on day 14. PCNA and PAX3 double-positive satellite cells were observed in the original rostral tissues, indicating the involvement of satellite cell proliferation during tail regeneration. Taken together, these data suggest that tail regeneration in Gekko japonicus involved rapid angiogenesis from the beginning to the day 10 and followed by capillary remodeling. The innervation of regenerated tail was significant on day 4 and increased gradually during regeneration, while the regenerated muscle tissues was obvious on day 14 after amputation.

L-leucine promotes axonal outgrowth and regeneration via mTOR activation.

Discovering safe and effective drugs that promote neuron regeneration is an essential strategy for the recovery of central nervous system injuries. In this study, we found that L-leucine, an essential amino acid obtained from both supplements and food sources, could dramatically boost axonal outgrowth and regeneration. First, the effects of L-leucine on neurons were evaluated by cell apoptosis, survival, and death assays, and the results showed no changes in these processes after treatment. By live cell imaging, L-leucine was found to remarkably increase axonal length and growth velocity after axotomy. We also verified that L-leucine enhanced p-mTOR/p-S6K activation in neurons by testing with an mTOR inhibitor, rapamycin. Thereafter, we investigated the effects of L-leucine on the spinal cord injury in vivo. A mouse model of spinal cord hemi-section was established, and L-leucine was administered by tail intravenous injection. Basso mouse scale values revealed that L-leucine could improve functional recovery after injury. It was also notable that L-leucine treatment promoted axon growth across chondroitin sulfate proteoglycan (CSPG) areas. Furthermore, we used CSPGs as inhibitory environmental cues and clarified that L-leucine significantly enhanced axonal outgrowth and regeneration by promoting p-mTOR and p-S6K activation. Therefore, our study is the first to report that L-leucine promotes axonal regeneration in vitro and in vivo and could be candidate drug for axonal re-growth and nervous functional recovery.

GIP-GIPR promotes neurite outgrowth of cortical neurons in Akt dependent manner.

The intrinsic capacity of axonal growth is varied among the neurons form different tissues or different developmental stages. In this study, we established an in vitro model to compare the axonal growth of neurons from embryonic 18 days, post-natal 1 day and post-natal 3 days rat. The E18 neurons showed powerful ability of neuritogenensis and axon outgrowth and the ability decreased rapidly along with development. The transcriptome profile of these neurons revealed a set of genes positively correlated with the capacity of neurite outgrowth. Glucose-dependent insulinotropic polypeptide receptor (GIPR) is identified as a gene to promote neurite outgrowth, which was approved by siRNA knock down assay in E18 neuron. Glucose-dependent insulinotropic polypeptide (GIP), a ligand of GIPR secreted from enteroendocrine K cells, is well-known for its role in nutrient sensing and intake. To verify the effect of GIP-GIPR signal on neurite outgrowth, we administrated GIP to stimulate the E18 neurons, the results showed that GIP significantly improved extension of axon. We further revealed that GIP increased Rac1/Cdc42 phosphorylation in Akt dependent manner. In summary, our study established an in vitro model to screen the genes involved in neurite outgrowth, and we provided mechanical insight on the GIP-GIPR axis to promote axonal outgrowth.

Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli.

The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNAUTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNAUTu and seven chimeric tRNAs that were constructed on the basis of tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNAUTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

Expression and characterization of recombinant bifunctional enzymes with glutathione peroxidase and superoxide dismutase activities.

To balance the production and decomposition of reactive oxygen species, living organisms have generated antioxidant enzymes and non-enzymatic antioxidant defense systems. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) are two important antioxidant enzymes. Apart from their catalytic functions, they protect each other, resulting in more efficient removal of reactive oxygen species, protection of cells against injury, and maintenance of the normal metabolism of reactive oxygen species. SOD catalyzes the dismutation of the superoxide anion (O2•-) to oxygen (O2) and hydrogen peroxide (H2O2). H2O2 is then detoxified to water by GPx. In this study, human GPx1Ser and the Alvinella pompejana SOD (ApSOD) gene were used to design and generate several recombinant proteins with both GPx and SOD activities by combining traditional fusion protein technology, a cysteine auxotrophic expression system, and a single protein production (SPP) system. Among the fusion proteins, Se-hGPx1Ser-L-ApSOD exhibited the highest SOD and GPx activities. Additional research was conducted to better understand the properties of Se-hGPx1Ser-L-ApSOD. The synergism of Se-hGPx1Ser-L-ApSOD was evaluated by using an in vitro model. This research may facilitate future studies on the cooperation and catalytic mechanisms of GPx and SOD. We believe that the bifunctional enzyme has potential applications as a potent antioxidant.

Heterologous expression and characterization of human cellular glutathione peroxidase mutants.

Cellular glutathione peroxidase (GPx1; EC1.11.1.9) is a major intracellular antioxidant selenoenzyme in mammals. However, the complicated expression mechanism of selenocysteine (Sec)-containing protein increases the difficulty of expressing human GPx1 (hGPx1) in Escherichia coli (E. coli). In this study, hGPx1 gene was cloned from a cDNA library of the human hepatoma cell line HepG2. The codon UGA encoding Sec49 of hGPx1 was first mutated to UGC encoding cysteine (Cys) and then biosynthetically converted to Sec during expression in an E. coli BL21(DE3)cys auxotrophic system. Seleno-GPx1Sec displayed a low GPx activity of 522 U/µmol. To improve the activity, the other five Cys residues (C2, C78, C115, C156, C202) were mutated to serine (Ser) in one hGPx1 molecule. The mutant seleno-hGPx1Ser showed a high activity of 5278 U/µmol, which was more than 10-fold enhanced as compared with seleno-GPx1Sec . The activity was the highest among all of those seleno-proteins obtained by this method so far. Kinetic analysis of seleno-hGPx1Ser showed a typical ping-pong mechanism, which was similar to those of natural GPxs. This research will be of value in overcoming the problem of limited sources of natural GPx and substantially promotes the research of the characterization of GPx. © 2014 IUBMB Life, 66(3):212-219, 2014.