Katsuhiko Hata

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA–Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.

Rho-ROCK Inhibitors as Emerging Strategies to Promote Nerve Regeneration

Several myelin-associated proteins in the central nervous system (CNS) have been identified as inhibitors of axonal regeneration following the injury of the adult vertebrate CNS. Among these inhibitors, myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp) are well characterized. Recently, the repulsive guidance molecule (RGM) was included as a potent myelin-derived neurite outgrowth inhibitor in vitro and in vivo. The discovery of the receptors and downstream signals of these inhibitors enabled further understanding of the mechanism underlying the failure of axonal regeneration. The activation of RhoA and its effector Rho kinases (ROCK) after the ligation of these inhibitors to the corresponding receptors has been shown to be a key element for axonal growth inhibition. Blockade of the Rho-ROCK pathway reverses the inhibitory effects of these inhibitors in vitro and promotes axonal regeneration in vivo. Therefore, the Rho-ROCK inhibitors have a therapeutic potential against injuries to the human CNS, such as spinal cord injuries.

Promotion of Axon Regeneration by Myelin-Associated Glycoprotein and Nogo through Divergent Signals Downstream of Gi/G

Several myelin-derived proteins have been identified as components of the CNS myelin that prevents axonal regeneration in the adult vertebrate CNS. Activation of RhoA has been shown to be an essential part of the signaling mechanism of these proteins. Here we report an additional signal, which determines whether these proteins promote or inhibit axon outgrowth. Myelin-associated glycoprotein (MAG) and Nogo trigger the intracellular elevation of Ca2+ as well as the activation of PKC, presumably mediated by Gi/G. Neurite outgrowth inhibition and growth cone collapse by MAG or Nogo can be converted to neurite extension and growth cone spreading by inhibiting conventional PKC, but not by inhibiting inositol 1,4,5-triphosphate (IP3). Conversely, neurite growth of immature neurons promoted by MAG is abolished by inhibiting IP3. Activation of RhoA is independent of PKC. Thus, a balance between PKC and IP3 is important for bidirectional regulation of axon regeneration by the myelin-derived proteins.

BMP inhibition enhances axonal growth and functional recovery after spinal cord injury

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor‐β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP‐2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5‐positive neurons

Netrin‐1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A–Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin‐1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine‐rich transmembrane protein‐3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5‐positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2‐expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ‐derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5‐positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Neogenin, a Receptor for Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) regulate many mammalian physiologic and pathophysiologic processes. These proteins bind with the kinase receptors BMPR-I and BMPR-II, thereby activating Smad transcription factor. In this study, we demonstrate that neogenin, a receptor for netrins and proteins of the repulsive guidance molecule family, is a receptor for BMPs and modulates Smad signal transduction. Neogenin was found to bind directly with BMP-2, BMP-4, BMP-6, and BMP-7. Knockdown of neogenin in C2C12 cells resulted in the enhancement of the BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1, Smad5, and Smad8. Conversely, overexpression of neogenin in C2C12 cells suppressed these processes. Our results also indicated that BMP-induced activation of RhoA was mediated by neogenin. Inhibition of RhoA promoted BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1/5/8. However, treatment with Y-27632, an inhibitor of Rho-associated protein kinase, did not modulate BMP-induced phosphorylation of Smad1/5/8. Taken together, our findings suggest that neogenin negatively regulates the functions of BMP and that this effect of neogenin is mediated by the activation of RhoA.

Limited functional recovery in rats with complete spinal cord injury after transplantation of whole-layer olfactory mucosa: laboratory investigation.

OBJECTnThe olfactory mucosa (OM) consists of 2 layers, the epithelium and the lamina propria. Attempts have been made to restore motor function in rat models of spinal cord injury (SCI) by transplanting olfactory ensheathing cells from the lamina propria, but there has been no attempt to transplant the OM in animal models. To investigate the potential of the OM to restore motor function, the authors developed a rat model of SCI and delayed transplantation of syngenic OM.nnnMETHODSnTwo weeks after complete transection of the spinal cord at the T-10 level in Wistar rats, pieces of syngenic whole-layer OM were transplanted into the lesion. Rats that underwent respiratory mucosa transplantation were used as controls. The authors evaluated the locomotor activity according to the Basso-Beattie-Bresnahan scale for 8 weeks after transplantation. Obtained spinal cords were analyzed histologically. Results The OM transplantation rats showed significantly greater hindlimb locomotor recovery than the respiratory mucosa-transplanted rats. However, the recovery was limited according to the Basso-Beattie-Bresnahan scale. In the histological examination, the serotonergic raphespinal tract was regenerated. The pseudocyst cavity volume in the vicinity of the SCI lesion correlated negatively with the functional recovery.nnnCONCLUSIONSnTransplantation of whole-layer OM in rats contributes to functional recovery from SCI, but the effect is limited. In addition to OM transplantation, other means would be necessary for better outcomes in clinical situations.

Neogenin regulates neuronal survival through DAP kinase

The repulsive guidance molecule (RGM) is a membrane-bound protein that has diverse functions in the developing central nervous system. Identification of neogenin as a receptor for RGM provided evidence of its cell death-inducing activity in the absence of RGM. Here, we show that the serine/threonine kinase death-associated protein kinase (DAPK) is involved in the signal transduction of neogenin. Neogenin interacts with DAPK and reduces DAPK autophosphorylation on Ser308 in vitro. Neogenin-induced cell death is abolished in the presence of RGM or by blocking DAPK. Although neogenin overexpression or RGM downregulation in the chick neural tube in vivo induces apoptosis, coexpression of the dominant-negative mutant or small-interference RNA of DAPK attenuates this proapoptotic activity. Thus, RGM/neogenin regulates cell fate by controlling the DAPK activity.

Zyxin is a novel interacting partner for SIRT1

BackgroundSIRT1 is a mammalian homologue of NAD+-dependent deacetylase sirtuin family. It regulates longevity in several model organisms and is involved with cell survival, differentiation, metabolism among other processes in mammalian cells. SIRT1 modulates functions of various key targets via deacetylation. Recent studies have revealed SIRT1 protects neurons from axonal degeneration or neurodegeneration. Further, SIRT1 null mice exhibit growth retardation and developmental defects, suggesting its critical roles in neurons and development.ResultsTo identify novel binding partners for SIRT1 in the central nervous system, we performed yeast two-hybrid screening on human fetal brain cDNA library and found that zyxin is a possible binding partner. SIRT1 and zyxin transcript were both preferentially expressed in developmental mouse brain. Zyxin accumulates in the nucleus where it is co-localized with SIRT1 after treatment with leptomycin B in COS-7 cells. Furthermore, SIRT1 deacetylates zyxin, suggesting SIRT1 could interact with nuclear-accumulated zyxin and modulate its function through deacetylation.ConclusionZyxin could be a novel interacting partner of SIRT1. Zyxin is an adaptor protein at focal adhesion plaque, regulating cytoskeletal dynamics and signal transduction to convey signal from the ECM (extracellular matrix) to the nucleus. Our results raise the possibility that SIRT1 regulates signal transmission from ECM to the nucleus by modulating the functions of zyxin via deacetylation.

Rho Kinase Inhibitor Improves Motor Dysfunction and Hypoalgesia in a Rat Model of Lumbar Spinal Canal Stenosis

Study Design. Immunohistochemical and behavioral study using a rat cauda equina compression model. Objective. To investigate, after cauda equina compression by spinal canal stenosis (SCS), Rho activation in the spinal cord and cauda equina, and the effect of intrathecal administration of a Rho kinase inhibitor on hypoalgesia and motor dysfunction. Summary of Background Data. Compression of the cauda equina caused by SCS is a common clinical disorder associated with sensory disturbance and intermittent claudication. Cauda equina compression is thought to reduce blood flow and result in nerve degeneration caused by various cytokines. Rho, a member of the small GTPases, is a signal transmitter. It promotes Wallerian degeneration, decreases blood flow in the spinal cord and brain, and increases expression of several cytokines. Currently, Rho kinase inhibitor is used clinically to treat progressive nerve damage due to cerebrovascular disorders. However, its effect for SCS has not been evaluated. Methods. Forty-two 6-week-old male Sprague-Dawley rats (200–250 g) were used. For the SCS model (n = 27), a small piece of silicon was placed under the lamina of the fourth lumbar vertebra. In the sham-operated group, laminectomies were performed at L5 only (n = 15). We examined mechanical sensitivity and motor function using von Frey hairs and a treadmill, and immunohistochemically localized Rho in the spinal ventral neurons, axons, and Schwann cells in the cauda equina. We also examined the effects of intrathecally administered Rho kinase inhibitor for hypoalgesia or motor dysfunction caused by SCS. Results. We observed motor dysfunction and hypoalgesia and activated Rho-immunoreactive cells in spinal ventral neuroreported to induce neurite and axonal outgrowth in the spinal cord and brain after nervous system injury.10,11 In addition, 1 report showed that Rho kinase was involved in Wallerian degeneration that was rescued by Rho kinase inhibitor.16 Furthermore, it is thought that Rho is involved in TNF-&agr; and interleukin (IL) production in the central nervous system, and the production was inhibited by administering Rho kinase inhibitor in the central nervous system.17 Regardns, axons, and Schwann cells in the cauda equina. Intrathecal administration of Rho kinase inhibitor improved mechanical hypoalgesia and motor dysfunction caused by SCS. Conclusion. Activated Rho may play an important role in nerve damage in the cauda equina in SCS. Rho kinase inhibitor may be a useful tool in determining the pathomechanism of cauda equina syndrome caused by SCS.