Toru Ogata

Opposing Extracellular Signal-Regulated Kinase and Akt Pathways Control Schwann Cell Myelination

Schwann cells are the myelinating glia of the peripheral nervous system, and their development is regulated by various growth factors, such as neuregulin, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). However, the mechanism of intracellular signaling pathways following these ligand stimuli in Schwann cell differentiation remains elusive. Here, we demonstrate that in cultured Schwann cells, neuregulin and PDGF suppressed the expression of myelin-associated protein markers, whereas IGF-I promoted it. Although these ligands activated common downstream signaling pathways [i.e., extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt pathways], the profiles of activation varied among ligands. To elucidate the function of these pathways and the mechanisms underlying Schwann cell differentiation, we used adenoviral vectors to selectively activate or inactivate these pathways. We found that the selective activation of Erk pathways suppressed Schwann cell differentiation, whereas that of PI3K pathways promoted it. Furthermore, lithium chloride, a modulator of glycogen synthase kinase-3β (GSK-3β) promoted Schwann cell differentiation, suggesting the involvement of GSK-3β as a downstream molecule of PI3K-Akt pathways. Selective activation of PI3K pathways in Schwann cells by gene transfer also demonstrated increased myelination in in vitro Schwann cell-DRG neuron cocultures and in vivo allogenic nerve graft experiments. We conclude that signals mediated by PI3K-Akt are crucial for initiation of myelination and that the effects of growth factors are primarily dependent on the balance between Erk and PI3K-Akt activation. Our results also propose the possibility of augmenting Schwann cell functions by modulating intracellular signals in light of future cell therapies.

Signaling axis in schwann cell proliferation and differentiation.

Recent progress in molecular biology has markedly expanded our knowledge of the molecular mechanism behind the proliferation and differentiation processes of Schwann cells, the myelinforming cells in peripheral nervous systems. Intracellular signaling molecules participate in integrating various stimuli from cytokines and other humoral factors and control the transcriptional activities of the genes that regulate mitosis or differentiation. This article provides an overview of the roles played by the intracellular pathways regulating Schwann cell functions. In Schwann cell proliferation, cyclic adenosine monophosphate signals and mitogen-activated protein kinase pathways is regulated by various cytokines and extracellular matrix molecules. Specifically, platelet-derived growth factor, neuregulin, and insulin-like growth factor-I all are classified as ligands for receptor-type tyrosine kinase and activate common intracellular signaling cascades, mitogen-activated protein kinase pathways, and phosphatidylinositol-3-kinase pathways. The balance of activities between these two pathways appears crucial in regulating Schwann cell differentiation, in which phosphatidylinositol-3-kinase pathways promote myelin formation. Analysis of these signaling molecules in Schwann cells will enable us not only to understand their physiological development but also to innovate new approaches to treat disorders related to myelination.

Phosphorylated neurofilament subunit NF-H as a biomarker for evaluating the severity of spinal cord injury patients, a pilot study

Study design:A pilot cross-sectional study of patients with acute cervical spinal cord injury (SCI).Objectives:The precise evaluation of the severity of SCI is important for developing novel therapies. Although several biomarkers in cerebrospinal fluid have been tested, few analyses of blood samples have been reported. A novel biomarker for axonal injury, phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), has been reported to be elevated in blood from rodent SCI model. The aim of this study is to investigate whether pNF-H values in blood can serve as a biomarker to evaluate the severity of patients with SCI.Setting:Tokyo Metropolitan Bokutoh Hospital and National Rehabilitation Center, Japan.Methods:This study enrolled 14 patients with acute cervical SCI. Sequential plasma samples were obtained from 6 h to 21 days after injury. Patients were classified according to American Spinal Injury Association impairment scale (AIS) at the end of the follow-up (average, 229.1 days). Plasma pNF-H values were compared between different AIS grades.Results:In patients with complete SCI, pNF-H became detectable at 12 h after injury and remained elevated at 21 days after injury. There was a statistically significant difference between AIS A (complete paralysis) patients and AIS C (incomplete paralysis) patients.Conclusions:Plasma pNF-H was elevated in accordance with the severity of SCI and reflected a greater magnitude of axonal damage. Therefore, pNF-H is a potential biomarker to independently distinguish AIS A patients (complete SCI) from AIS C–E patients (incomplete SCI). However, further studies are required to evaluate its utility in predicting prognosis of patients in the incomplete category.

In vivo messenger RNA introduction into the central nervous system using polyplex nanomicelle.

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.

Akt and PP2A Reciprocally Regulate the Guanine Nucleotide Exchange Factor Dock6 to Control Axon Growth of Sensory Neurons

The phosphorylation status of Dock6 causes sensory neuron axons to switch from extension to branching. Switching from Axon Extension to Branching Understanding the signaling pathways that enable neurons, such as dorsal root ganglion neurons, to extend axons into the periphery can identify potential therapeutic targets to promote nerve regeneration after injury. Miyamoto et al. found that the guanine nucleotide exchange factor Dock6, which regulates the cytoskeleton, was required for axonal extension during development and after nerve crush injury. Phosphorylation of Dock6 by the kinase Akt inhibited its activity; dephosphorylation by the phosphatase PP2A promoted Dock6 activity. During development, peripheral sensory neurons extend axons until the axons contact their targets, at which point they branch. During developmental stages when axons were growing, extending periods were associated with the interaction of PP2A with Dock6 and low Dock6 phosphorylation, whereas periods of branching were associated with the interaction of Akt with Dock6 and high Dock6 phosphorylation. Rescue experiments indicated that the phosphorylation status of Dock6 switched dorsal root ganglion neurons from axon extension to branching. Thus, regulation of the phosphorylation status of Dock6 by Akt and PP2A determines whether it promotes axon extension or branching in dorsal root ganglion neurons, and timing of treatments targeting this pathway for nerve regeneration is essential to proper reinnervation. During neuronal development, axons navigate long distances, eventually forming precise connections with such targets as peripheral tissues. Dock6 is a guanine nucleotide exchange factor (GEF) that activates the Rho family guanosine triphosphatases Rac1 and Cdc42 to regulate the actin cytoskeleton. We found that phosphorylation of Ser1194 in Dock6 inhibited its GEF activity and suppressed axonal growth of embryonic sensory neurons and axon regeneration of postnatal sensory neurons in vitro and in vivo. At early developmental stages, when axons are growing, the protein phosphatase PP2A interacted with and dephosphorylated Dock6, thereby increasing the activity of Dock6. At later developmental stages, the abundance of the kinase Akt increased, resulting in the binding of Akt to Dock6 and the phosphorylation of Dock6 at Ser1194. In dorsal root ganglion neurons from mice lacking Dock6, reintroduction of Dock6 with a nonphosphorylatable S1194A mutation rescued axon extension but not branch number, whereas reintroduction of Dock6 with a phosphomimetic S1194E mutation resulted in premature branching. Thus, the phosphorylation status of Dock6 at Ser1194 determines whether it promotes axon extension or branching in sensory neurons, revealing interplay between kinase and phosphatase action on a Rho-GEF during axon growth.

Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts

The role of TGF-beta/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector-mediated gene transduction system. Expression of constitutively active activin receptor-like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.

Lipopolysaccharide preconditioning facilitates M2 activation of resident microglia after spinal cord injury

The inflammatory response following spinal cord injury (SCI) has both harmful and beneficial effects; however, it can be modulated for therapeutic benefit. Endotoxin/lipopolysaccharide (LPS) preconditioning, a well‐established method for modifying the immune reaction, has been shown to attenuate damage induced by stroke and brain trauma in rodent models. Although such effects likely are conveyed by tissue‐repairing functions of the inflammatory response, the mechanisms that control the effects have not yet been elucidated. The present study preconditioned C57BL6/J mice with 0.05 mg/kg of LPS 48 hr before inducing contusion SCI to investigate the effect of LPS preconditioning on the activation of macrophages/microglia. We found that LPS preconditioning promotes the polarization of M1/M2 macrophages/microglia toward an M2 phenotype in the injured spinal cord on quantitative real‐time polymerase chain reaction, enzyme‐linked immunosorbent assay, and immunohistochemical analyses. Flow cytometric analyses reveal that LPS preconditioning facilitates M2 activation in resident microglia but not in infiltrating macrophages. Augmented M2 activation was accompanied by vascularization around the injured lesion, resulting in improvement in both tissue reorganization and functional recovery. Furthermore, we found that M2 activation induced by LPS preconditioning is regulated by interleukin‐10 gene expression, which was preceded by the transcriptional activation of interferon regulatory factor (IRF)−3, as demonstrated by Western blotting and an IRF‐3 binding assay. Altogether, our findings demonstrate that LPS preconditioning has a therapeutic effect on SCI through the modulation of M1/M2 polarization of resident microglia. The present study suggests that controlling M1/M2 polarization through endotoxin signal transduction could become a promising therapeutic strategy for various central nervous system diseases.

Association between in vivo knee kinematics during gait and the severity of knee osteoarthritis

BACKGROUND Osteoarthritis patients may exhibit different kinematics according to the disease stage. However, changes in the frontal and horizontal planes in each stage remain unclear. The purpose of this study was to investigate changes in the knee kinematic gait variables of osteoarthritis patients, including the frontal and horizontal planes, with respect to the severity of the disease. METHODS Forty-five patients with knee osteoarthritis and 13 healthy young subjects were recruited for the experiment. All subjects were examined while walking on a 10-m walkway at a self-selected speed. In each trial, we calculated the angular displacements of flexion/extension, abduction/adduction, and external/internal tibial rotation. We also measured muscle strength, range of motion (ROM), and alignment. We compared the differences in osteoarthritis severity and knee kinematic variables between osteoarthritis patients and normal subjects. RESULTS The flexion angle at the time of foot contact was significantly less in patients with severe and moderate osteoarthritis than in normal subjects (both p<0.01). The abduction angle at the 50% stance phase was significantly less in patients with severe osteoarthritis than in normal subjects (p<0.05). The excursion of axial tibial rotation was significantly less in patients with early osteoarthritis than in normal subjects (p<0.05). CONCLUSION Osteoarthritis patients had different knee kinematics during gait, depending on the progress of osteoarthritis. Early-stage patients exhibit decreased axial tibial rotation excursion, while severe-stage patient exhibit increased knee adduction.

Amputee locomotion: Spring-like leg behavior and stiffness regulation using running-specific prostheses

Carbon fiber running-specific prostheses (RSPs) have allowed individuals with lower extremity amputation (ILEA) to participate in running. It has been established that as running speed increases, leg stiffness (Kleg) remains constant while vertical stiffness (Kvert) increases in able-bodied runners. The Kvert further depends on a combination of the torsional stiffnesses of the joints (joint stiffness; Kjoint) and the touchdown joint angles. Thus, an increased understanding of spring-like leg function and stiffness regulation in ILEA runners using RSPs is expected to aid in prosthetic design and rehabilitation strategies. The aim of this study was to investigate stiffness regulation to various overground running speeds in ILEA wearing RSPs. Eight ILEA performed overground running at a range of running speeds. Kleg, Kvert and Kjoint were calculated from kinetic and kinematic data in both the intact and prosthetic limbs. Kleg and Kvert in both the limbs remained constant when running speed increased, while intact limbs in ILEA running with RSPs have a higher Kleg and Kvert than residual limbs. There were no significant differences in Kankle, Kknee and touchdown knee angle between the legs at all running speeds. Hip joints in both the legs did not demonstrate spring-like function; however, distinct impact peaks were observed only in the intact leg hip extension moment at the early stance phase, indicating that differences in Kvert between limbs in ILEA are due to attenuating shock with the hip joint. Therefore, these results suggest that ILEA using RSPs has a different stiffness regulation between the intact and prosthetic limbs during running.

Hyperphosphorylated neurofilament NF-H as a biomarker of the efficacy of minocycline therapy for spinal cord injury

Study design:An in vivo study in a rat model of acute spinal cord contusion.Objectives:To assess the efficacy of novel therapies for acute spinal cord injury (SCI), methods to evaluate accurately the effects of these therapies should be developed. Although neurological examination is commonly used for this purpose, unstable clinical conditions and the spontaneous recovery of neurological function in the acute and subacute phases after injury make this measurement unreliable. Recent studies have reported that the phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), a new biomarker for axonal degeneration, can be measured in serum samples in experimental SCI animals. Therefore, we aimed to investigate the use of plasma pNF-H as an indicator of the efficacy of minocycline, a neuroprotective drug, for treating SCI.Setting:This study was carried out at Saitama, Japan.Methods:Spinal cord injured rats received either minocycline or saline intraperitoneally. The plasma pNF-H levels and functional hind limb score were determined after the injury.Results:Minocycline treatment reduced plasma pNF-H levels at 3 and 4 days post-injury (dpi). Rats with lower plasma pNF-H levels at 3 dpi had higher hind limb motor score at 28 dpi.Conclusions:pNF-H levels may serve as a biomarker for evaluating the efficacy of therapies for SCI.